Enhancing the therapeutic activity of immune checkpoint inhibitor

ABSTRACT

The present invention provides antagonists and methods of use thereof in the treatment of cancer and abnormal immune suppression diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/435,425, filed Feb. 17, 2017 (now U.S. Pat. No. 10,906,977), andclaims the benefit of priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/296,808, filed Feb. 18, 2016, each ofwhich is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersCA091645, HL065301, HL083151, RR015555, GM103392, and RR018789, awardedby the National Institutes of Health, and grant number W81XWH-14-1-0178,awarded by the Department of the Army. The government has certain rightsin the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The content of the text file named“48420-513001US_Sequence_Listing_ST25.txt,” which was created on Sep.17, 2020 and is 8 KB in size, is hereby incorporated herein by referencein its entirety.

BACKGROUND OF THE INVENTION

Extracellular matrix (ECM) remodeling regulates angiogenesis. However,prior to the invention described herein, the precise mechanisms by whichstructural changes in ECM proteins contribute to angiogenesis were notfully understood. The role of integrin, e.g., integrin αvβ3 or integrinα10β1, in angiogenesis is complex, as evidence exists for both positiveand negative functions. As such, prior to the invention describedherein, there was a pressing need to develop an understanding of therole of integrin in angiogenesis.

SUMMARY OF THE INVENTION

The invention is based, in part, upon the surprising discovery that anantagonist (i.e., Mab XL313) specifically directed to a cryptic RGDKGE(SEQ ID NO: 1)-containing collagen epitope significantly enhanced theanti-tumor efficacy of an antibody that targets the immune checkpointregulatory protein, PDL-1. Additionally, the invention is based, inpart, on an antagonist of integrin αvβ3 that is used to enhance thetherapeutic activity of an immune checkpoint inhibitor, e.g., cytotoxicT-lymphocyte-associated protein 4 (CTLA-4), programmed death ligand-1(PDL-1 or PD-L1), programmed cell death protein 1 (PD-1),lymphocyte-activation gene 3 (Lag3), leukocyte-associatedimmunoglobulin-like receptor 1 (LAIR1) and/or leukocyte-associatedimmunoglobulin-like receptor 2 (LAIR2) antibodies. The invention is alsobased, in part, upon the surprising discovery that an antagonist (i.e.,Mab HU177) specifically directed to a cryptic CPGFPGFC (SEQ ID NO:16)-containing collagen epitope enhanced the anti-tumor efficacy of anantibody that targets the immune checkpoint regulatory protein, PDL-1.The invention is also based, in part, upon the discovery that reducingthe expression of an integrin receptor that can serve as a cell surfacereceptor for the HU177 epitope (i.e., integrin α10β1) can reduceexpression of a potent immunosuppressive cytokine (i.e., IL-10) inmelanoma cells in vitro. Conversely, as described in detail below,injecting a soluble version of the HU177 epitope itself inducesexpression of a second potent immunosuppressive molecule, TGF-β, inmouse serum in vivo.

The present invention features compositions and methods for treatingcancer and diseases characterized by abnormal immune suppression. Forexample, methods of treating cancer in a subject are carried out byidentifying a subject, e.g., a human subject, that has been diagnosedwith cancer; administering an immune checkpoint inhibitor; andadministering an antagonist of collagen or a fragment thereof, therebytreating cancer in the subject. In some cases, the immune checkpointinhibitor comprises an inhibitor of CTLA-4, PDL-1, PD-1, CTLA-4, Lag3,LAIR1 and/or LAIR2. For example, the inhibitor of PDL-1 comprises aPDL-1 antibody.

Suitable types of collagen include collagen type-I, collagen type-II,collagen type-III and collagen type-IV (e.g., the alpha 6 chain ofcollagen type-IV). In some cases, the antagonist of collagen or afragment thereof comprises an antagonist of the XL313 cryptic collagenepitope or an antagonist of the HU177 cryptic collagen epitope. Forexample, the antagonist of the XL313 cryptic collagen epitope comprisesan antibody that binds a cryptic RGDKGE (SEQ ID NO: 1) containingcollagen epitope or wherein the antagonist of the HU177 cryptic collagenepitope comprises an antibody that binds a cryptic CPGFPGFC (SEQ ID NO:16)-containing collagen epitope. Preferably, the antibody comprises amonoclonal antibody, e.g., an XL313 monoclonal antibody or an HU177monoclonal antibody.

Preferably, the antagonist of collagen or a fragment thereof enhancesanti-tumor activity of the immune checkpoint inhibitor and inhibits aninflammatory condition. Exemplary inflammatory conditions includedermatitis, pneumonitis, or colitis.

The methods described herein can be used in conjunction with one or morechemotherapeutic or anti-neoplastic agents. In some cases, theadditional chemotherapeutic agent is radiotherapy. In some cases, thechemotherapeutic agent is a cell death-inducing agent.

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

Exemplary cancers are selected from the group comprising of melanoma,central nervous system (CNS) cancer, CNS germ cell tumor, lung cancer,leukemia, multiple myeloma, renal cancer, malignant glioma,medulloblatoma, breast cancer, ovarian cancer, prostate cancer, bladdercancer, fibrosarcoma, pancreatic cancer, gastric cancer, head and neckcancer, colorectal cancer. For example, a cancer cell is derived from asolid cancer or hematological cancer. The hematological cancer is, e.g.,a leukemia or a lymphoma. A leukemia is acute lymphoblastic leukemia(ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia(CML), or acute monocytic leukemia (AMoL). A lymphoma is follicularlymphoma, Hodgkin's lymphoma (e.g., Nodular sclerosing subtype,mixed-cellularity subtype, lymphocyte-rich subtype, or lymphocytedepleted subtype), or Non-Hodgkin's lymphoma. Exemplary solid cancersinclude but are not limited to melanoma (e.g., unresectable, metastaticmelanoma), renal cancer (e.g., renal cell carcinoma), prostate cancer(e.g., metastatic castration resistant prostate cancer), ovarian cancer(e.g., epithelial ovarian cancer, such as metastatic epithelial ovariancancer), breast cancer (e.g., triple negative breast cancer), and lungcancer (e.g., non-small cell lung cancer).

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice or nonhuman primates, as a skilledartisan recognizes it is routine in the art to modify the dosage forhumans compared to animal models. In certain embodiments, it isenvisioned that the dosage of the antagonist to collagen may vary frombetween about 0.1 μg compound/kg body weight to about 25000 μgcompound/kg body weight; or from about 1 μg/kg body weight to about 4000μg/kg body weight or from about 10 μg/kg body weight to about 3000 μg/kgbody weight. In other embodiments this dose may be about 0.1, 0.3, 0.5,1, 3, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000,2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,8500, 9000, 9500, 10000, 10500, 1100, 11500, 12000, 12500, 13000, 13500,14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500,19000, 19500, 20000, 20500, 21000, 21500, 22000, 22500, 23000, 23500,24000, 24500, or 25000 μg/kg body weight. In other embodiments, it isenvisaged that doses may be in the range of about 0.5 μg compound/kgbody weight to about 20 μg compound/kg body weight. In otherembodiments, the doses may be about 0.5, 1, 3, 6, 10, or 20 mg/kg bodyweight. Of course, this dosage amount may be adjusted upward ordownward, as is routinely done in such treatment protocols, depending onthe results of the initial clinical trials and the needs of a particularpatient.

In some cases, the immune checkpoint inhibitor, e.g., the inhibitor ofPDL-1, is administered at a dosage of 0.01-10 mg/kg (e.g., 0.01, 0.05,0.1, 0.5, 1, 5, or 10 mg/kg) bodyweight. For example, the PDL-1inhibitor is administered in an amount of 0.01-30 mg (e.g., 0.01, 0.05,0.1, 0.5, 1, 5, 10, 20, or 30 mg) per dose. In another example, theimmune checkpoint inhibitor, e.g., the anti-PD-L1 antibody, isadministered in the dose range of 0.1 mg/kg to 10 mg/kg of body weight.In some cases, the XL313 antibody or the HU177 antibody is administeredat a dosage of 0.01-10 mg/kg (e.g., 0.01, 0.05, 0.1, 0.5, 1, 5, or 10mg/kg) bodyweight. For example, the XL313 antibody or the HU177 antibodyis administered in an amount of 0.01-30 mg (e.g., 0.01, 0.05, 0.1, 0.5,1, 5, 10, 20, or 30 mg) per dose. For example, the dose range of MabX1313 or Mab HU177 is from 0.1 mg/kg to 25 mg/kg of body weight.

The compositions of the invention (e.g., inhibitor of PDL-1, XL313antibody, and HU177 antibody) are administered once per month, twice permonth (i.e., every two weeks), every week, once per day, twice per day,every 12 hours, every 8 hours, every 4 hours, every 2 hours or everyhour. The compositions of the invention (e.g., inhibitor of PDL-1, XL313antibody, and HU177 antibody) are administered for a duration of 1 day,2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4weeks, five weeks, six weeks, 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2years, 3 years, 4 years, 5 years or more. For example, the compositionof the invention (e.g., inhibitor of PD-L1, XL313 antibody, and HU177antibody) are administered one dose every two weeks for 4 to 6 weeks oruntil the disease is treated.

Also provided is a method of treating a disease characterized byabnormal immune suppression in a subject by identifying a subject, e.g.,a human, that has been diagnosed with a disease characterized byabnormal immune suppression, administering an immune checkpointinhibitor, and administering an antagonist of an integrin, therebytreating in the subject.

Suitable immune checkpoint inhibitors comprise an inhibitor of CTLA-4,PD-1, PDL-1, Lag3, LAIR1, or LAIR 2. For example, the immune checkpointinhibitor comprises a CTLA-4 antibody, a PD-1 antibody, a PDL-1antibody, a Lag3 antibody, a LAIR1 antibody, or a LAIR 2 antibody.

Preferably, the integrin comprises integrin αvβ3. For example, theantagonist of integrin αvβ3 comprises an antibody targeting αvβ3 bindingRGDKGE (SEQ ID NO: 1) containing collagen epitope. Alternatively, theintegrin comprises integrin α10β1. For example, the antagonist ofintegrin α10β1 comprises an antibody targeting α10β1 binding CPGFPGFC(SEQ ID NO: 16)-containing collagen epitope.

The methods described herein can be used in conjunction with one or morechemotherapeutic or anti-neoplastic agents. In some cases, theadditional chemotherapeutic agent is radiotherapy. In some cases, thechemotherapeutic agent is a cell death-inducing agent.

Suitable diseases characterized by abnormal immune suppression includeType I diabetes, lupus, psoriasis, scleroderma, hemolytic anemia,vasculitis, Graves' disease, rheumatoid arthritis, multiple sclerosis,Hashimoto's thyroiditis, Myasthenia gravis, and vasculitis

In some cases, the immune checkpoint inhibitor (e.g., CTLA-4 antibody, aPD-1 antibody, a PDL-1 antibody, Lag3, LAIR1, or LAIR 2) is administeredat a dosage of 0.01-10 mg/kg (e.g., 0.01, 0.05, 0.1, 0.5, 1, 5, or 10mg/kg) bodyweight. For example, the PDL-1 inhibitor is administered inan amount of 0.01-30 mg (e.g., 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, or 30mg) per dose. In another example, the antibody is administered in thedose range of 0.1 mg/kg to 10 mg/kg of body weight. In some cases, theantagonist of integrin αvβ3 is administered at a dosage of 0.01-10 mg/kg(e.g., 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 mg/kg) bodyweight. In somecases, the XL313 antibody or the HU177 antibody is administered in anamount of 0.01-30 mg (e.g., 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, or 30mg) per dose.

The compositions of the invention (e.g., immune checkpoint inhibitor andantagonist of integrin αvβ3) are administered once per month, twice permonth (once every two weeks), once a week, once per day, twice per day,every 12 hours, every 8 hours, every 4 hours, every 2 hours or everyhour. The compositions of the invention (e.g., inhibitor of PDL-1, XL313antibody, and HU177 antibody) are administered for a duration of 1 day,2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4years, 5 years or more. The composition of the invention (e.g.,inhibitor of PDL-1. XL313 antibody, and HU177 antibody) are administeredone dose every two weeks for 4 to 6 weeks or until the disease istreated

Also provided is a method of treating a disease characterized by anoveractive immune response (e.g., an autoimmune disease) in a subject,e.g., a human subject, that has been diagnosed with an overactive immuneresponse by administering a peptide comprising collagen or a fragmentthereof, thereby treating overactive immune response in the subject.Suitable types of collagen include collagen type-I, collagen type II,collagen type III, and collagen type-IV (e.g., the alpha 6 chain ofcollagen type-IV). For example, the peptide comprises RGDKGE (SEQ IDNO: 1) or CPGFPGFC (SEQ ID NO: 16).

In some cases, the autoimmune disease comprises Graves' disease,Hashimoto's thyroiditis, Systemic lupus erythematosus (lupus), Type 1diabetes, multiple sclerosis or rheumatoid arthritis.

Methods for healing a wound in a subject, e.g., a human subject with awound, are carried out by administering a peptide comprising collagen ora fragment thereof to the wound of the subject, thereby healing a woundin the subject. For example, the peptide is administered to a site thatis about 0.1 mm, 0.5 mm, 1 mm, 2.5 mm, 5 mm, 10 mm, 15 mm, 20 mm, or 40mm away from a perimeter or margin of the wound. Alternatively, thepeptide is administered directly to the wound itself.

Suitable types of collagen include collagen type-I, collagen type II,collagen type III, and collagen type-IV (e.g., the alpha 6 chain ofcollagen type-IV). For example, the peptide comprises RGDKGE (SEQ IDNO: 1) or CPGFPGFC (SEQ ID NO: 16).

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “antibody” is meant any immunoglobulin polypeptide, or fragmentthereof, having immunogen binding ability. As used herein, the term“antibodies” includes polyclonal antibodies, affinity-purifiedpolyclonal antibodies, monoclonal antibodies, and antigen-bindingfragments, such as F(ab′)₂ and Fab proteolytic fragments. Geneticallyengineered intact antibodies or fragments, such as chimeric antibodies,Fv fragments, single chain antibodies, and the like, as well assynthetic antigen-binding peptides and polypeptides, are also included.Non-human antibodies may be humanized by grafting non-human CDRs ontohuman framework and constant regions, or by incorporating the entirenon-human variable domains. In certain preferred embodiments, humanizedantibodies may retain non-human residues within the human variableregion framework domains to enhance proper binding characteristics.

“Antigenic fragment” and the like are understood as at least thatportion of a peptide capable of inducing an immune response in asubject, or being able to be specifically bound by an antibody raisedagainst the antigenic fragment. Typically, antigenic fragments are atleast 7 amino acids in length. Antigenic fragments can include deletionsof the amino acid sequence from the N-terminus or the C-terminus, orboth. For example, an antigenic fragment can have an N- and/or aC-terminal deletion of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, or more amino acids. Antigenicfragments can also include one or more internal deletions of the sameexemplary lengths. Antigenic fragments can also include one or morepoint mutations, particularly conservative point mutations. At least anantigenic fragment of protein can include the full length, wild-typesequence of the antigen. An antigenic fragment can include more than onepotential antibody binding site. An antigenic fragment can be used tomake antibodies for use in any of the methods provided herein.

By “autoimmune disease” is meant a disease characterized by adysfunction in the immune system. The disease is characterized by thecomponents of the immune system affected, whether the immune system isoveractive or underactive, or whether the condition is congenital oracquired. In most cases, the disorder causes abnormally low activity orover activity of the immune system. In cases of immune system overactivity, the body attacks and damages its own tissues (autoimmune).Immune deficiency diseases decrease the body's ability to fightinvaders, causing vulnerability to infections. In response to an unknowntrigger, the immune system may begin producing antibodies that insteadof fighting infections, attack the body's own tissues. Treatment forautoimmune diseases generally focuses on reducing immune systemactivity.

By “blood vessel formation” is meant the dynamic process that includesone or more steps of blood vessel development and/or maturation, such asangiogenesis, arteriogenesis, vasculogenesis, formation of an immatureblood vessel network, blood vessel remodeling, blood vesselstabilization, blood vessel maturation, blood vessel differentiation, orestablishment of a functional blood vessel network.

By “blood vessel remodeling” or “vascular remodeling” is meant thedynamic process of blood vessel enlargement in shape and size tomaintain the luminal orifice and blood flow. For example, vascularremodeling includes change in arterial size to adapt to plaqueaccumulation, effectively maintaining the lumen and blood flow to themyocardium.

As used herein, “binding” or “specific binding” is understood as havingat least a 10³ or more, preferably 10⁴ or more, preferably 10⁵ or more,preferably 10⁶ or more preference for binding to a specific bindingpartner as compared to a non-specific binding partner (e.g., binding anantigen to a sample known to contain the cognate antibody).

By “cancer” is meant, comprising of but not limited to melanoma, centralnervous system (CNS) cancer, CNS germ cell tumor, lung cancer, leukemia,multiple myeloma, renal cancer, malignant glioma, medulloblatoma, breastcancer, ovarian cancer, prostate cancer, bladder cancer, fibrosarcoma,pancreatic cancer, gastric cancer, head and neck cancer, colorectalcancer. For example, a cancer cell is derived from a solid cancer orhematological cancer. The hematological cancer is, e.g., a leukemia or alymphoma. A leukemia is acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma (SLL), chronic myelogenous leukemia (CML), or acutemonocytic leukemia (AMoL). A lymphoma is follicular lymphoma, Hodgkin'slymphoma (e.g., Nodular sclerosing subtype, mixed-cellularity subtype,lymphocyte-rich subtype, or lymphocyte depleted subtype), orNon-Hodgkin's lymphoma. Exemplary solid cancers include but are notlimited to melanoma (e.g., unresectable, metastatic melanoma), renalcancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastaticcastration resistant prostate cancer), ovarian cancer (e.g., epithelialovarian cancer, such as metastatic epithelial ovarian cancer), breastcancer (e.g., triple negative breast cancer), and lung cancer (e.g.,non-small cell lung cancer).

By “control” or “reference” is meant a standard of comparison. As usedherein, “changed as compared to a control” sample or subject isunderstood as having a level of the analyte or diagnostic or therapeuticindicator to be detected at a level that is statistically different thana sample from a normal, untreated, or control sample. Control samplesinclude, for example, cells in culture, one or more laboratory testanimals, or one or more human subjects. Methods to select and testcontrol samples are within the ability of those in the art. An analytecan be a naturally occurring substance that is characteristicallyexpressed or produced by the cell or organism (e.g., an antibody, aprotein) or a substance produced by a reporter construct (e.g,β-galactosidase or luciferase). Depending on the method used fordetection the amount and measurement of the change can vary.Determination of statistical significance is within the ability of thoseskilled in the art, e.g., the number of standard deviations from themean that constitute a positive result.

By “cryptic” is meant that a motif may be inaccessible to cell surfacereceptors, and once the target protein is proteolyzed or denatured, asequence becomes exposed or generates a fragment that is then recognizedby the antibody. For example, the XL313 epitope, i.e., RGDKGE (SEQ IDNO: 1) core sequence within collagen type-I is cryptic in that theantibody does not react with normal collagen in its triple helicalstate, but once it is proteolyzed or denatured, the sequence becomesexposed or generates a fragment of collagen that is recognized by MabXL313. Similarly, the HU177 epitope, i.e., CPGFPGFC (SEQ ID NO: 16) coresequence within collagen type-I is cryptic in that the antibody does notreact with normal collagen in its triple helical state, but once it isproteolyzed or denatured, the sequence becomes exposed or generates afragment of collagen that is recognized by Mab HU177.

As used herein, “detecting”, “detection” and the like are understoodthat an assay performed for identification of a specific analyte in asample, e.g., an antigen in a sample or the level of an antigen in asample. The amount of analyte or activity detected in the sample can benone or below the level of detection of the assay or method.

By “diagnosing” and the like as used herein refers to a clinical orother assessment of the condition of a subject based on observation,testing, or circumstances for identifying a subject having a disease,disorder, or condition based on the presence of at least one indicator,such as a sign or symptom of the disease, disorder, or condition.Typically, diagnosing using the method of the invention includes theobservation of the subject for multiple indicators of the disease,disorder, or condition in conjunction with the methods provided herein.Diagnostic methods provide an indicator that a disease is or is notpresent. A single diagnostic test typically does not provide adefinitive conclusion regarding the disease state of the subject beingtested.

By the terms “effective amount” and “therapeutically effective amount”of a formulation or formulation component is meant a sufficient amountof the formulation or component, alone or in a combination, to providethe desired effect. For example, by “an effective amount” is meant anamount of a compound, alone or in a combination, required to amelioratethe symptoms of a disease relative to an untreated patient. Theeffective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

The term “polynucleotide” or “nucleic acid” as used herein designatesmRNA, RNA, cRNA, cDNA or DNA. As used herein, a “nucleic acid encoding apolypeptide” is understood as any possible nucleic acid that upon(transcription and) translation would result in a polypeptide of thedesired sequence. The degeneracy of the nucleic acid code is wellunderstood. Further, it is well known that various organisms havepreferred codon usage, etc. Determination of a nucleic acid sequence toencode any polypeptide is well within the ability of those of skill inthe art.

As used herein, “immunoassay” is understood as any antibody basedetection method including, but not limited to enzyme linkedimmunosorbent assay (ELISA), radioimmune assay (RIA), Western blot,immunohistochemistry, immunoprecipitation assay such as LuciferaseImmunoprecipitation System (LIPS see, e.g., US Patent Publication2007/0259336 which is incorporated herein by reference). In a preferredembodiment, the immunoassay is a quantitative. Antibodies for use inimmunoassays include any monoclonal or polyclonal antibody appropriatefor use in the specific immunoassay.

By “inhibitory nucleic acid molecule” is meant a polynucleotide thatdisrupts the expression of a target nucleic acid molecule or an encodedpolypeptide. Exemplary inhibitory nucleic acid molecules include, butare not limited to, shRNAs, siRNAs, antisense nucleic acid molecules,and analogs thereof.

As used herein, “isolated” or “purified” when used in reference to apolypeptide means that a naturally polypeptide or protein has beenremoved from its normal physiological environment (e.g., proteinisolated from plasma or tissue, optionally bound to another protein) oris synthesized in a non-natural environment (e.g., artificiallysynthesized in an in vitro translation system or using chemicalsynthesis). Thus, an “isolated” or “purified” polypeptide can be in acell-free solution or placed in a different cellular environment (e.g.,expressed in a heterologous cell type). The term “purified” does notimply that the polypeptide is the only polypeptide present, but that itis essentially free (about 90-95%, up to 99-100% pure) of cellular ororganismal material naturally associated with it, and thus isdistinguished from naturally occurring polypeptide. Similarly, anisolated nucleic acid is removed from its normal physiologicalenvironment. “Isolated” when used in reference to a cell means the cellis in culture (i.e., not in an animal), either cell culture or organculture, of a primary cell or cell line. Cells can be isolated from anormal animal, a transgenic animal, an animal having spontaneouslyoccurring genetic changes, and/or an animal having a genetic and/orinduced disease or condition. An isolated virus or viral vector is avirus that is removed from the cells, typically in culture, in which thevirus was produced.

As used herein, “kits” are understood to contain at least onenon-standard laboratory reagent for use in the methods of the inventionin appropriate packaging, optionally containing instructions for use.The kit can further include any other components required to practicethe method of the invention, as dry powders, concentrated solutions, orready to use solutions. In some embodiments, the kit comprises one ormore containers that contain reagents for use in the methods of theinvention; such containers can be boxes, ampules, bottles, vials, tubes,bags, pouches, blister-packs, or other suitable container forms known inthe art. Such containers can be made of plastic, glass, laminated paper,metal foil, or other materials suitable for holding reagents.

As used herein, “obtaining” is understood herein as manufacturing,purchasing, or otherwise coming into possession of.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations.

Formulations of the present invention include those suitable for oral,nasal, topical, transdermal, buccal, sublingual, intramuscular,intracardiac, intraperotineal, intrathecal, intracranial, rectal,vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient that can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound thatproduces a therapeutic effect.

In some cases, a composition of the invention is administered orally orsystemically. Other modes of administration include rectal, topical,intraocular, buccal, intravaginal, intracisternal,intracerebroventricular, intratracheal, nasal, transdermal, within/onimplants, or parenteral routes. The term “parenteral” includessubcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal,or infusion. Intravenous or intramuscular routes are not particularlysuitable for long-term therapy and prophylaxis. They could, however, bepreferred in emergency situations. Compositions comprising a compositionof the invention can be added to a physiological fluid, such as blood.Oral administration can be preferred for prophylactic treatment becauseof the convenience to the patient as well as the dosing schedule.Parenteral modalities (subcutaneous or intravenous) may be preferablefor more acute illness, or for therapy in patients that are unable totolerate enteral administration due to gastrointestinal intolerance,ileus, or other concomitants of critical illness. Inhaled therapy may bemost appropriate for pulmonary vascular diseases (e.g., pulmonaryhypertension).

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

As used herein, “plurality” is understood to mean more than one. Forexample, a plurality refers to at least two, three, four, five, or more.

A “polypeptide” or “peptide” as used herein is understood as two or moreindependently selected natural or non-natural amino acids joined by acovalent bond (e.g., a peptide bond). A peptide can include 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturalor non-natural amino acids joined by peptide bonds. Polypeptides asdescribed herein include full length proteins (e.g., fully processedproteins) as well as shorter amino acids sequences (e.g., fragments ofnaturally occurring proteins or synthetic polypeptide fragments).Optionally the peptide further includes one or more modifications suchas modified peptide bonds, i.e., peptide isosteres, and may containamino acids other than the 20 gene-encoded amino acids. The polypeptidesmay be modified by either natural processes, such as posttranslationalprocessing, or by chemical modification techniques which are well knownin the art. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature. Modifications can occur anywhere in a polypeptide, includingthe peptide backbone, the amino acid side-chains and the amino orcarboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Also, a given polypeptide may contain manytypes of modifications. Polypeptides may be branched, for example, as aresult of ubiquitination, and they may be cyclic, with or withoutbranching. Cyclic, branched, and branched cyclic polypeptides may resultfrom posttranslation natural processes or may be made by syntheticmethods. Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formulation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, Proteins, Structure and Molecular Properties, 2nd ed., T. E.Creighton, W.H. Freeman and Company, New York (1993); PosttranslationalCovalent Modification of Proteins, B. C. Johnson, ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol 182:626-646(1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

The term “reduce” or “increase” is meant to alter negatively orpositively, respectively, by at least 5%. An alteration may be by 5%,10%, 25%, 30%, 50%, 75%, or even by 100%.

As used herein, a “reporter protein” or a “reporter polypeptide” isunderstood as a polypeptide that can be readily detected, preferablyquantitatively detected, either directly or indirectly. A reporterpolypeptide typically has an enzymatic activity, luciferase activity,alkaline phosphatase activity, beta-galactosidase activity, acetyltransferase activity, etc. wherein catalysis of a reaction with thesubstrate by the enzyme results in the production of a product, e.g.,light, a product that can be detected at a specific wavelength of light,radioactivity, such that the amount of the reporter peptide can bedetermined in the sample, either as a relative amount, or as an absoluteamount by comparison to control samples.

A “sample” as used herein refers to a biological material that isisolated from its environment (e.g., blood or tissue from an animal,cells, or conditioned media from tissue culture) and is suspected ofcontaining, or known to contain an analyte, such as a protein. A samplecan also be a partially purified fraction of a tissue or bodily fluid. Areference sample can be a “normal” sample, from a donor not having thedisease or condition fluid, or from a normal tissue in a subject havingthe disease or condition. A reference sample can also be from anuntreated donor or cell culture not treated with an active agent (e.g.,no treatment or administration of vehicle only). A reference sample canalso be taken at a “zero time point” prior to contacting the cell orsubject with the agent or therapeutic intervention to be tested or atthe start of a prospective study.

“Sensitivity and specificity” are statistical measures of theperformance of a binary classification test. The sensitivity (alsocalled recall rate in some fields) measures the proportion of actualpositives which are correctly identified as such (e.g. the percentage ofsick people who are identified as having the condition); and thespecificity measures the proportion of negatives which are correctlyidentified (e.g. the percentage of well people who are identified as nothaving the condition). They are closely related to the concepts of typeI and type II errors. A theoretical, optimal prediction can achieve 100%sensitivity (i.e. predict all people from the sick group as sick) and100% specificity (i.e. not predict anyone from the healthy group assick).

The concepts are expressed mathematically as follows:

sensitivity=#true positives/#true positives+#false negatives

specificity=#true negatives/#true negatives+#false positives.

By “selectively” is meant the ability to affect the activity orexpression of a target molecule without affecting the activity orexpression of a non-target molecule.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

A “subject” as used herein refers to an organism. In certainembodiments, the organism is an animal. In certain embodiments, thesubject is a living organism. In certain embodiments, the subject is acadaver organism. In certain preferred embodiments, the subject is amammal, including, but not limited to, a human or non-human mammal. Incertain embodiments, the subject is a domesticated mammal or a primateincluding a non-human primate. Examples of subjects include humans,monkeys, dogs, cats, mice, rats, cows, horses, goats, and sheep. A humansubject may also be referred to as a patient.

A “subject sample” can be a sample obtained from any subject, typicallya blood or serum sample, however the method contemplates the use of anybody fluid or tissue from a subject. The sample may be obtained, forexample, for diagnosis of a specific individual for the presence orabsence of a particular disease or condition.

A subject “suffering from or suspected of suffering from” a specificdisease, condition, or syndrome has a sufficient number of risk factorsor presents with a sufficient number or combination of signs or symptomsof the disease, condition, or syndrome such that a competent individualwould diagnose or suspect that the subject was suffering from thedisease, condition, or syndrome. Methods for identification of subjectssuffering from or suspected of suffering from conditions associated withdiminished cardiac function is within the ability of those in the art.Subjects suffering from, and suspected of suffering from, a specificdisease, condition, or syndrome are not necessarily two distinct groups.

As used herein, “susceptible to” or “prone to” or “predisposed to” aspecific disease or condition and the like refers to an individual whobased on genetic, environmental, health, and/or other risk factors ismore likely to develop a disease or condition than the generalpopulation. An increase in likelihood of developing a disease may be anincrease of about 10%, 20%, 50%, 100%, 150%, 200%, or more.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference. Genbank and NCBI submissions indicated by accessionnumber cited herein are incorporated herein by reference. All otherpublished references, documents, manuscripts and scientific literaturecited herein are incorporated herein by reference. In the case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1F are bar graphs of cryptic RGD containing peptides fromcollagen type-I support cell adhesion. Peptides corresponding to the 5different RGD-containing motifs of human collagen type-I weresynthesized with flanking cysteines and immobilized non-tissue cultureplates. The ability of stromal cells to attach (FIG. 1A-FIG. 1D) ormonoclonal antibodies to bind the immobilized peptides (FIG. 1E-FIG. 1F)was assessed. FIG. 1A is a bar graph that depicts human umbilical veinendothelial cell (HUVEC) adhesion to immobilized peptides. FIG. 1B is abar graph that depicts human retinal microvascular endothelial cell(HMVEC) adhesion to immobilized peptides. FIG. 1C is a bar graph thatdepicts HRMVEC adhesion to immobilized peptides. FIG. 1D is a bar graphthat depicts human dermal fibroblast (HDF) adhesion to immobilizedpeptides. FIG. 1E is a bar graph that depicts reactivity of Mab XL313 toimmobilized peptides. FIG. 1F is a bar graph that depicts reactivity ofMab XL166 to immobilized peptides. Data bars represent mean binding ±SDfrom at least 3 experiments each using triplicate wells. *P<0.05.

FIG. 2A-FIG. 2D is a series of immunoblots and bar graphs depicting thatMab XL313 exhibits selective binding to proteolyzed collagen type-I.Purified collagen type-I and collagen type-IV were incubated withcontrol buffer or activated MMP-2 over a time course and analyzed byWestern blot (FIG. 2A, FIG. 2B and FIG. 2D) or ELISA (FIG. 2C). FIG. 2Ais a Western blot depicting analysis of control not treatment (NT) andMMP-2 proteolyzed (Prot) collagen type-I (Left panel) and collagentype-IV (Right panel) probed with anti-collagen type-I specific antibody(Left panel) or anti-collagen type-IV specific antibody (Right panel).FIG. 2B is a Western blot depicting analysis of control non-proteolyzedand MMP-2 proteolyzed collagen type-I and type-IV following 16 hourincubation and probed with Mab XL313 directed to the RGDKGE (SEQ IDNO: 1) containing collagen motif. FIG. 2C is a bar graph depicting thedetection of Mab XL313 reactive RGDKGE (SEQ ID NO: 1) motif innon-proteolyzed (Nat) or MMP-2 proteolyzed (Prot) collagen by ELISA.Data bars indicate mean reactivity from triple wells ±SD. *P<0.05.Experiments were completed at least 3 times with similar results. FIG.2D is a Western blot depicting the analysis of the time dependentgeneration of the Mab XL313 reactive epitope of collagen type-1.

FIG. 3A-FIG. 3E are data depicting the differential roles of cryptic RGDcollagen motifs on angiogenesis and inflammation in vivo. Chickchorioallantoic membranes (CAMs) were either un-stimulated or stimulatedwith FGF-2 in the presence or absence of cortisone acetate. FIG. 3Ashows images of representative examples of un-stimulated or FGF-2stimulated CAM tissues stained with Mabs XL313 and XL166 (Green). Scalebars represent 50 m. FIG. 3B is a bar graph depicting the quantificationof FGF-2 induced angiogenesis in the absence or presence of Mabs XL313,XL166 or non-specific control antibody. Data bars represent mean numberof angiogenic branching vessels ±S.E from 8-10 animals per conditions.Experiments were completed at least 3 times with similar results. FIG.3C shows images of un-stimulated or FGF-2 stimulated CAMs in the absenceof cortisone acetate. Representative examples of inflammatory CAMthickening (Top panel), giemsa positive inflammatory infiltrates (Middlepanel) and infiltration of macrophages (green) following staining withanti-avian specific macrophage marker KUL1 (Bottom Panel). Scale barsrepresent 50 μm. FIG. 3D is a bar graph depicting the quantification ofrelative macrophage infiltration following FGF-2 stimulation in theabsence of cortisone acetate. Data bars represent mean±S.E levels of KULexpressing avian macrophages per 100× microscopic field from 5 fieldsper CAM and 10 CAM per condition. *P<0.05. FIG. 3E is a bar graphdepicting the quantification of FGF-2 induced inflammation in theabsence or presence of Mabs XL313, XL166 or non-specific controlantibody. Data bars represent mean percentage inflammatory CAMs ±S.E.Experiments were completed at least 3 times with similar results.*P<0.05.

FIG. 4A-FIG. 4D are images depicting generation of XL313 cryptic RGDKGE(SEQ ID NO: 1) epitope. FIG. 4A is a Western blot of whole cell lysates(Right panel) or serum free conditioned medium (Left panel) from stromalcells probed with Mab XL313 or loading control antibodies for 13-Actinor MMP-9. FIG. 4B are images of representative examples of FGF-2stimulated CAM tissues co-stained for expression of avian macrophages(Green) and the RGDKGE (SEQ ID NO: 1) containing epitope (Red). Scalebars indicate 50 μm. FIG. 4C is a Western blot of whole cell lysates(Right panel) or serum free conditioned medium (Left panel) frommacrophage cell lines probed with Mab XL313 or loading controlantibodies for β-Actin or IGFBP-4. FIG. 4D is a Western blot of wholecell lysates or serum free conditioned medium from RAW 264.7 macrophagecell that were either not transfected (WT) or transfected with α2(I)specific shRNA (Col 1 Kd) or control non-specific shRNA (Cont Kd) withMab XL313 (Left panel) or anti-collagen-1 antibody (right).

FIG. 5A-FIG. 5D are bar graphs depicting the induction of angiogenesisand inflammation in vivo by soluble RGDKGE (SEQ ID NO: 1) but not arelated RGDAPG (SEQ ID NO: 11) containing collagen epitope. FIG. 5A is abar graph depicting serum from chick embryos in which the CAMs had beeneither not stimulated (NT) or stimulated with FGF-2 was collected.Relative levels of circulating Mab XL313 reactive epitope werequantified by solid-phase ELISA. Data represent mean Mab XL313reactivity ±S.E (N=12 to 13). FIG. 5B is a bar graph depictingquantification of dose dependent induction of angiogenesis by RGDKGE(SEQ ID NO: 1) containing collagen peptide P-2. Data bars indicate meannumber of branch points ±S.E from 8 to 12 animal per condition. FIG. 5Cis a bar graph depicting quantification of CAM angiogenesis associatedwith RGD containing collagen peptides P-1 (KGDRGDAPG; SEQ ID NO: 2), P-2(QGPRGDKGE; SEQ ID NO: 3) or control peptide P-C (QGPSGSPGE; SEQ ID NO:4). Data bars indicate mean angiogenic index derived by subtracting thenumber of branch points from non-stimulated CAMs ±S.E from 8 to 12animals per condition. FIG. 5D is a bar graph depicting thequantification of CAM inflammation associated with RGD containingcollagen peptides P-1 (KGDRGDAPG; SEQ ID NO: 2), P-2 (QGPRGDKGE; SEQ IDNO: 3) or control peptide P-C (QGPSGSPGE (SEQ ID NO: 4)). Allexperiments were conducted at least 3 times with similar results.*P<0.05.

FIG. 6A-FIG. 6F are data depicting RGDKGE (SEQ ID NO: 1) containingcollagen peptide P-2 induced angiogenesis depends on P38MAPK.Angiogenesis was induced within the chick CAMs and the relative level ofP38MAPK was assessed. FIG. 6A is a Western blot of examples (N=3 percondition) of total lysate from un-stimulated (NT) or CAMs stimulationwith P-1 (KGDRGDAPG (SEQ ID NO: 2)) or P-2 (QGPRGDKGE (SEQ ID NO: 3))and probed for phosphorylated P38MAPK (P-p38), total P38MAPK (T-p38) orβ-Actin. FIG. 6B is a bar graph depicting the quantification (Image-J)of the mean relative levels of phosphorylated P38MAPK from un-stimulatedor following CAM stimulation with P-1 (KGDRGDAPG (SEQ ID NO: 2); SEQ IDNO: 2) or P-2 (QGPRGDKGE; SEQ ID NO: 3). Data bars represent mean levelsof phosphorylated P38MAPK±S.D (N=8 to 10 CAMs per condition). FIG. 6Care images of representative examples of P-2 stimulated CAM tissuesco-stained for expression of vWf and phosphorylated P38MAPK. Scale barindicates 50.0 μm. FIG. 6D is a Western blot analysis of endothelialcell (HUVEC) lysates from cells attached to collagen peptides P-1(KGDRGDAPG; SEQ ID NO: 2), P-2 (QGPRGDKGE; SEQ ID NO: 3) and probed forphosphorylated P38MAPK (P-p38), total P38MAPK (T-p38) or β-Actin. FIG.6E is a Western blot analysis of endothelial cell (HRMVEC) lysates fromcells attached to collagen peptides P-1 (KGDRGDAPG (SEQ ID NO: 2)), P-2(QGPRGDKGE (SEQ ID NO: 3)) and probed for phosphorylated P38MAPK(P-p38), total P38MAPK (T-p38) or β-Actin. FIG. 6F is a bar graphdepicting the quantification of collagen peptide P-2 (QGPRGDKGE (SEQ IDNO: 3)) induced angiogenesis in the chick CAM following treatment withP38MAPK inhibitor (P38IN) of DMSO control (Cont). Data bars representangiogenic vessel branch points ±S.E (N=8-12 per condition). Experimentswere conducted at least 3 times with similar results. *P<0.05.

FIG. 7A-FIG. 7G are images depicting collagen peptide P-2 binds andactivates αvβ3 and stimulates P38MAPK activation in a Src dependentmanner. FIG. 7A and FIG. 7B are bar graphs depicting that endothelialcells (HUVECS) were allowed to bind to wells coated with either RGDKGE(SEQ ID NO: 1) containing collagen peptide P-2 (FIG. 7A) or RGDAPG (SEQID NO: 11) containing collagen peptide P-1 (FIG. 7B) in the presence orabsence of function blocking anti-integrin antibodies. Data barsrepresent mean cell adhesion (±S.E. from 4 experiments). *p<0.05. FIG.7C is a Western blot of HUVEC lysates following binding to RGDcontaining collagen peptides P-1 or P-2 probed with anti-phosphorylatedβ3-integrin (p-β3), total β3-integrin (T-β3) or β-Actin. FIG. 7D is aWestern blot of HUVEC lysates following binding to RGD containingcollagen peptides P-1 or P-2 probed with anti-phosphorylated Src(β-Src), total Src (T-Src) or β-Actin. FIG. 7E is a Western blot oflysates prepared from HUVECs incubated in the presence or absence of aSrc inhibitor PP2 (Src-IN) or control DMSO (cont) following binding toRGD containing collagen peptides P-1 or P-2 probed withanti-phosphorylated β3-integrin (p-β3), total β3-integrin (T-β3) orβ-Actin. FIG. 7F is a Western blot of lysates prepared from HUVECsincubated in the presence or absence of a Src inhibitor PP2 (Src-IN) orcontrol DMSO (cont) following binding to RGD containing collagenpeptides P-1 or P-2 probed with anti-phosphorylated P38MAPK (p-P38),total P38MAPK-(T-P38) or β-Actin. FIG. 7G are images of representativeexamples of endothelial cells (HUVECs) attached to RGD containingcollagen peptides P-1 and P-2 stained for F-actin. Scale bar indicates50.0 μm.

FIG. 8A-FIG. 8I are data depicting that cellular interactions withRGDKGE (SEQ ID NO: 1) collagen-containing peptide accelerates actinstress fiber formation and nuclear YAP accumulation promotingendothelial growth. FIG. 8A are images of representative examples ofendothelial cells (HUVECs) attached to RGD containing collagen peptidesP-1 and P-2 stained for YAP (green). Scale bar indicates 50.0 μm. FIG.8B is a Western blot of cytoplasmic (left) and nuclear (right) fractionsof HUVEC lysates following binding to RGD containing collagen peptidesP-1 or P-2 probed with anti-YAP, β-tubulin and anti-total bindingprotein (TBP). FIG. 8C and FIG. 8D are bar graphs depicting thequantification of HUVEC (FIG. 8C) and HRMVEC (FIG. 8D) growth in thepresence or absence of exogenously added RGD containing peptides P-1 orP-2. Data bars represent mean endothelial cell growth (±S.E) from 3 to 4independent experiments expressed as percent of control. FIG. 8E is aWestern blot of isolated nuclear fractions from cell lysates ofendothelial cells (HRMVEC) following stimulation with soluble RGDcontaining collagen peptides P-1 or P2 and probed with YAP or totalbinding protein (TBP). FIG. 8F is a Western blot of isolated nuclearfractions from cell lysates of endothelial cells (HRMVEC) pre-incubatedwith Src or P38MAPK inhibitor and stimulation with soluble RGDcontaining collagen peptides P-1 or P-2 and probed with YAP or totalbinding protein (TBP). FIG. 8G and FIG. 8H are bar graphs depicting thequantification of endothelial cell growth in the presence or absence ofexogenously added RGD containing peptides P-1 or P-2 from cellstransduced with non-specific shRNA (FIG. 8G) or YAP specific shRNA (FIG.8H). Data bars represent mean endothelial cell growth (±S.E) from 4independent experiments. FIG. 8I is a bar graph depicting thequantification of endothelial cell growth in the presence or absence ofexogenously added VEGF (50 ng/ml) or 10% serum from cells transducedwith YAP specific shRNA. Data bars represent mean endothelial cellgrowth (±S.E) from 4 independent experiments. *P<0.05.

FIG. 9 is a bar graph depicting that Mab XL313 enhances the anti-tumoractivity of the immune checkpoint inhibitor for anti-PDL-1 antibody.Mice (C57BL/6) were injected with 3.5×10⁵ B16F10 melanoma cells. Micewere allowed to establish pre-existing tumors for 5 days prior totreatment. Mice were treated (100 μg/mouse) 3 times per week for 14days. Data represents mean tumor volume at day 14±SE from 8 mice percondition. P<0.05 was considered significant.

FIG. 10 are images depicting that enhanced expression of PDL-1 in humanM21 melanoma tumors as compared to M21L melanoma tumors lack αvβ3. Mice(nude) were injected with either M21 (αvβ3+) or M21L (αvβ3−) melanomacells. Mice were allowed to establish pre-existing tumors. Mice weresacrificed and tumors dissected and snap frozen. Tumor sections wereanalyzed for expression of the immune checkpoint regulatory proteinPDL-1 by immunofluorescence staining. Red color indicates expression ofPDL-1. Control indicates staining with secondary antibody only.

FIG. 11 are images depicting enhanced expression of PDL-1 in human M21melanoma tumors as compared to M21 melanoma tumors in which β3 integrinwas knocked down. M21 melanoma cells that express integrin αvβ3 weretransfected with non-specific control shRNA (M21 Cont) or β3 specificshRNA (M21β3 Kd). Mice (nude) were injected with either M21 Cont (αvβ3+)or M21β3 Kd (αvβ3−) melanoma cells. Mice were allowed to establishpre-existing tumors. Mice were sacrificed and tumors dissected and snapfrozen. Tumor sections were analyzed for expression of the immunecheckpoint regulatory protein PDL-1 by immunofluorescence staining. Redcolor indicates expression of PDL-1. Control indicates stainingsecondary antibody only.

FIG. 12 are images depicting reduced expression of PDL-1 in Melanomatumors in mice treated with an antibody targeting the αvβ3 ligand(RGDKGE (SEQ ID NO: 1) containing XL313 collagen epitope). Mice(C57BL/6) were injected with 3.5×10⁵ B16F10 melanoma cells. Mice wereallowed to establish pre-existing tumors for 5 days prior to treatment.Mice were treated (100 μg/mouse) 3 times a week for 14 days with anantibody targeting the αvβ3 binding RGDKGE (SEQ ID NO: 1) containingcollagen epitope. Mice were sacrificed and tumors dissected and snapfrozen. Tumor sections were analyzed for expression of the immunecheckpoint regulatory protein PDL-1 by immunofluorescence staining. Redcolor indicates expression of PDL-1. Control indicates staining withsecondary antibody only.

FIG. 13 are images depicting enhanced detection of lymphocyticinfiltrates in melanoma tumors in mice treated with an antibodytargeting the αvβ3 ligand (RGDKGE (SEQ ID NO: 1) containing XL313collagen epitope). Mice (C57BL/6) were injected with 3.5×10⁵ B16F10melanoma cells. Mice were allowed to establish pre-existing tumors for 5days prior to treatment. Mice were treated (100 μg/mouse) 3 times a weekfor 14 days with an antibody targeting the αvβ3 binding RGDKGE (SEQ IDNO: 1) containing collagen epitope. Mice were sacrificed and tumorsdissected. Tumor sections were analyzed for expression of the immunecheckpoint regulatory protein PDL-1 by Giemsa staining. Black arrowsindicate examples of areas with enhanced lymphocytic infiltrates.

FIG. 14A-FIG. 14C are blots depicting detection of enhanced levels ofPDL-1 protein in melanoma and endothelial cells following binding toαvβ3 ECM ligand (denatured collagen-IV). FIG. 14A and FIG. 14B are blotsof integrin αvβ3 expressing melanoma cells (M21 and CLL-49) and FIG. 14Cis a blot depicting that endothelial cells (HUVEC) were allowed toattach to well coated with either a non-αvβ3 binding ligand (nativecollagen-IV) or an αvβ3 binding ligand (denatured collagen-IV). Wholecell lysates were prepared and the relative levels of PDL-1 or loadingcontrol β-actin were assessed by Western blot.

FIG. 15 is a blot depicting enhanced levels of PDL-1 protein in melanomacells following binding to αvβ3 ligand XL313 epitope (RGDKGE (SEQ ID NO:1)). Integrin αvβ3 expressing melanoma cells (M21) were seeded oncontrol uncoated wells or wells immobilized with the XL313 crypticcollagen epitope (RGDKGE (SEQ ID NO: 1)). Following a 15 minuteincubation cell lysates were prepared. The relative levels of PDL-1 orloading control β-actin were assessed by Western blot.

FIG. 16 is a blot depicting a reduction in the levels of PDL-1 proteinin melanoma cells following blocking binding to αvβ3 ECM ligand(denatured collagen-IV) with a αvβ3 specific antibody LM609. Integrinαvβ3 expressing melanoma cells (M21) were mixed with a controlnon-specific (normal mouse Ig) or αvβ3 specific antibody (Mab LM609) andadded to wells coated with the αvβ3 binding ligand (denaturedcollagen-IV). Whole cell lysates were prepared following a 24 hourincubation period and the relative levels of PDL-1 or loading controlβ-actin were assessed by Western blot.

FIG. 17A-FIG. 17D show the generation of the HU177 epitope in ovariantumors. Biopsies from frozen sections of malignant human ovarian tumorsor benign granulomas were stained with Hematoxylin and Eosin (H&E) orwith anti-HU177 antibody. FIG. 17A shows examples of H&E staining (leftpanel) or staining for the HU177 cryptic collagen epitope (right panel)in malignant ovarian tumors. FIG. 17B shows examples of H&E staining(left panel) or staining for the HU177 cryptic collagen epitope (rightpanel) in benign granulomas. FIG. 17C shows the quantification of therelative levels of HU177 epitope within malignant ovarian tumors orbenign granulomas. Data bars represent the mean relative levels of HU177epitope+S.E from five 200× fields. *P<0.05 as compared to controls. FIG.17D shows examples of HU177 epitope (red) within SKOV-3 tumors, ornormal ovaries, lungs and liver. Photos were at 200×. Scale bar 63microns.

FIG. 18A-FIG. 18C show the inhibition of tumor growth by targeting theHU177 epitope. FIG. 18A and FIG. 18B shows graphs wherein SKOV-3 cellswere seeded topically on the CAMs of chick embryos. Twenty-four hourslater, the animals were untreated (NT) or treated once topically withanti-HU177 (100 ug) or control antibody (100 ug). FIG. 18A showsexamples of tumors from each condition. FIG. 18B shows thequantification of mean tumor weights from each condition following a7-day incubation period. Data bars represent mean tumor weights+S.E from7 to 8 animals per condition. FIG. 18C is a graph wherein mice wereinjected subcutaneously in the flanks with SKOV-3 cells and 5 days laterwere untreated or treated i.p (100 ug) with anti-HU177 antibody orcontrol antibody 3 times per week for 28 days. Data points representmean tumor volume+SE from 5 to 6 animals per condition. *P<0.05 ascompared to controls.

FIG. 19A-FIG. 19D show the analysis of SKOV-3 tumors growing in mice.Mice were injected subcutaneously in the flanks with SKOV-3 cells andfollowing a 5-day incubation period were untreated or treated i.p withanti-HU177 (100 ug) or control antibodies (100 ug) 3 times per week for28 days. FIG. 19A shows the analysis of the relative levels of Ki67expression per 200× field+SE in SKOV-3 tumors. FIG. 19B (Top) shows anexample of co-expression of CD-31 (green) and HU177 epitope (red) inuntreated SKOV-3 tumors. FIG. 19B (Bottom) shows an example ofco-expression of aSMA (red) and HU177 epitope (green) in untreatedSKOV-3 tumors. FIG. 19C is a photomicrograph wherein mice were injectedsubcutaneously in the flanks with SKOV-3 cells and following a 5 dayincubation period were either untreated or treated i.p with 100 ug ofanti-HU177 or control antibody 3 times per week for 28 days. FIG. 19C(Top panels) show examples of tumors from each condition stained forCD-31 (red). FIG. 19C (Bottom panel) show quantification of tumorangiogenesis with data bars representing mean vessels per 100× field+SE.FIG. 19D is a photomicrograph wherein mice were injected subcutaneouslyin the flanks with SKOV-3 cells and following a 5 day incubation periodwere either untreated or treated i.p with 100 ug of anti-HU177 orcontrol antibody 3 times per week for 28 days. FIG. 19D (Top panels)show examples of tumors from each condition stained for aSMA (red). FIG.19D (Bottom panel) shows quantification aSMA expressing infiltratingstromal cells with data bars representing mean aSMA expression per 100×field+SE. Scale bar 126 microns. *P<0.05 as compared to controls.

FIG. 20A-FIG. 20D is a series of bar graphs showing that SKOV-3 tumorcells and fibroblast exhibit enhanced adhesion and migration ondenatured collagen. Non-tissue culture wells and membranes fromtranswell migration chambers were coated with 5.0 ug/ml of nativecollagen (Nat-Coll) or denatured collagen (Den-Coll). SKOV-3 cells (FIG.20A and FIG. 20C) or fibroblasts (FIG. 20B and FIG. 20D) were allowed toattach or migrate on either native or denatured collagen. FIG. 20A is abar graph showing the quantification of mean SKOV-3 cell adhesion. Databars represent mean cell adhesion+SE from 4 independent experimentsexpressed as percent of control with adhesion on native collagen set at100%. FIG. 20B is a bar graph showing the quantification of meanfibroblast cell adhesion. Data bars represent mean cell adhesion+SE from4 independent experiments expressed as percent of control with adhesionon native collagen set at 100%. FIG. 20C is a bar graph showing thequantification of mean SKOV-3 cell migration. Data bars represent meancell migration+SE from 3 independent experiments expressed as percent ofcontrol with migration on native collagen set at 100%. FIG. 20D is a bargraph showing the quantification of mean fibroblast cell migration. Databars represent mean cell migration+SE from 4 independent experimentsexpressed as percent of control with migration on native collagen set at100%. *P<0.05 as compared to controls.

FIG. 21A-FIG. 21H show that blocking the HU177 epitope inhibits basaladhesion and migration on denatured collagen. Non-tissue culture wellsand membranes from transwell migration chambers were coated with 5.0ug/ml of native collagen or denatured collagen. SKOV-3 cells (FIG. 21A,FIG. 21B, FIG. 21E and FIG. 21F) or fibroblasts (FIG. 21C, FIG. 21D,FIG. 21G and FIG. 21H) were allowed to attach or migrate on eithernative or denatured collagen in the presence or absence of anti-HU177antibody or control antibody (50 ug/ml). FIG. 21A and FIG. 21B are bargraphs that show quantification of SKOV-3 cell adhesion to denatured ornative collagen in the presence or absence of anti-HU177 or controlantibody. Data bars represent mean cell adhesion indicated as percent ofcontrol+SE from 4 to 5 independent experiments with no treatment set at100%. FIG. 21C and FIG. 21D are bar graphs that show quantification offibroblast cell adhesion to denatured or native collagen in the presenceor absence of anti-HU177 or control antibody. Data bars represent meancell adhesion indicated as percent of control+SE from 3 to 7 independentexperiments with no treatment set at 100%. FIG. 21E and FIG. 21F are bargraphs showing quantification of SKOV-3 cell migration on denatured ornative collagen in the presence or absence of anti-HU177 or controlantibody. Data bars represent mean cell migration indicated as percentof control+SE from 4 independent experiments with no treatment set at100%. FIG. 21G and FIG. 21H are bar graphs showing the quantification offibroblast cell migration on denatured or native collagen in thepresence or absence of anti-HU177 or control antibody. Data barsrepresent mean cell migration indicated as percent of control+SE from 3to 4 independent experiments with no treatment set at 100%. *P<0.05 ascompared to controls.

FIG. 22A-FIG. 22E are a series of bar charts and an immunoblot showingthat targeting the HU177 epitope inhibits growth factor inducedmigration and Erk phosphorylation. Fibroblasts were resuspended in thepresence or absence of SKOV-3 CM (FIG. 22A) or FGF-2 (FIG. 22B) in thepresence or absence of anti-HU177 or control antibodies (50 ug/ml). FIG.22A and FIG. 22B are bar charts showing the quantification offibroblasts cell migration on denatured collagen. Data bars representmean migration indicated as percent of control+SE from 3 independentexperiments. FIG. 22C is a photograph showing Western blot analysis ofErk in lysates from fibroblasts seeded on denatured collagen in thepresence or absence of anti-HU177 or control antibody (50 ug/ml). FIG.22D is a bar chart showing the quantification of mean FGF2-induced Erkphosphorylation in the presence or absence of anti-HU177 antibody orcontrol antibody from fibroblasts seeded on denatured collagen from 4independent experiments. FIG. 22E is a bar chart wherein fibroblastswere resuspended in the presence or absence of FGF2 and in the presenceor absence of Mek inhibitor and allowed to migrate on denaturedcollagen. Data bars indicate mean cell migration indicated as percent ofcontrol+SE from 3 independent experiments. *P<0.05 as compared tocontrols.

FIG. 23A-FIG. 23G show that integrin α10β1 binds the HU177 collagenepitope. Wells were coated with denatured collagen (FIG. 23A, and FIG.23C-FIG. 23E), integrins α10β1 and α3β1 (FIG. 23B) or the syntheticHU177 epitope peptide or control peptide (FIG. 23F) and integrins (FIG.23A, FIG. 23C-FIG. 23E) or denatured collagen (FIG. 23B) and allowed tobind.

FIG. 23A is a line graph showing integrin binding to denatured collagen.FIG. 23B is a line graph showing binding of denatured collagen toimmobilized integrins. FIG. 23C is a bar graph showing binding of α10β1to denatured collagen in the presence or absence of anti-HU177 orcontrol antibodies. FIG. 23D is a bar graph showing the binding of α2β1to denatured collagen in the presence or absence of anti-HU177 orcontrol antibodies. FIG. 23E is a bar graph showing the binding of αVβ3to denatured collagen in the presence or absence of anti-HU177 orcontrol antibodies. FIG. 23F is a bar graph showing integrin binding tosynthetic HU177 epitope or control peptide. FIG. 23G is a bar graphshowing integrin binding to denatured collagen in the presence ofsynthetic HU177 epitope or control peptide. Experiments were completedat least 3 times. *P<0.05 as compared to controls.

FIG. 24A-FIG. 24I is a series of photomicrographs, immunoblots, and barcharts showing the expression and migratory function of α10β1. FIG. 24Ais a photomicrograph showing an example of expression of α10β1 (green)in human ovarian tumor biopsy (left) and SKOV-3 tumors (right). FIG. 24Bis a photomicrograph of an example of coexpression of α10β1 (green) andaSMA (red) expressing stromal cells within SKOV-3 tumors. Photos were at200×. FIG. 24C is an analysis of α10β1 expression in SKOV-3 cells orfibroblasts (HF) by western blot. FIG. 24D is a bar graph showing thequantification of FGF-2-induced fibroblasts migration in the presence orabsence of anti-α10β1 antibody or control. Data bars represent meanmigration indicated as percent of control+SE from 3 independentexperiments. FIG. 24E is a Western blot analysis of relative expressionof α10 integrin, α1 integrin or β-actin in cell lysates fromnon-specific control knock down fibroblasts (Con-Kd-HF) or α10 integrinknock down fibroblasts (α10-Kd-HF). FIG. 24F is a bar chart showing thequantification of FGF-2-induced migration of wild type (WT-HF), α10integrin knock down fibroblasts (α10-Kd-HF) or non-specific controlknock down fibroblasts (Con-Kd-HF) on denatured collagen. Data barsrepresent mean migration indicated as percent of control+SE from 3independent experiments. FIG. 24G is a bar chart of SKOV-3 cellproliferation in the presence of control or fibroblast CM. Data barsrepresent mean O.D 490 nm+SE. FIG. 24H is a Western blot analysis forKi67 in lysates of SKOV-3 cells treated with fibroblast CM. FIG. 24I isa bar chart showing SKOV-3 proliferation in the presence or absence offibroblast CM and in the presence of anti-IL-6 or control antibodies.Data bars represent mean O.D 490 nm+SE. All experiments were completedat least 3 times with similar results. Scale bar 63 microns. *P<0.05 ascompared to controls.

FIG. 25A-FIG. 25D is a series of bar charts showing that the HU177collagen epitope is recognized by integrin α10β1 which regulatesexpression of the immune suppressive cytokine IL-10. FIG. 25A is a barchart wherein a peptide (cPG) containing the HU177 collagen epitopeCPGFPGFC (SEQ ID NO: 16) was immobilized on microtiter wells and theability of recombinant integrin receptors to bind was analyzed by ELISA.Data bars represent mean integrin binding to the HU177 collagen epitope.FIG. 25B-FIG. 25D is a series of bar charts showing that the expressionof α10 integrin chain in human C8161 melanoma cells was reduced bytransfection of shRNA directed to the α10 integrin chain. Twenty-fourhour serum free conditioned medium from each melanoma variant wasconcentrated 10× and analyzed for the relative levels of cytokines. FIG.25B is a bar graph wherein data bars represent relative levels of IL-10as determined by ELISA. FIG. 25C is a bar graph wherein data barsrepresent relative levels of IL-2 as determined by ELISA. FIG. 25D is abar graph wherein data bars represent relative levels of IL-4 asdetermined by ELISA.

FIG. 26A-FIG. 26C is a series of bar charts showing that the injectionof a peptide (cPG) containing the HU177 collagen epitope inducesexpression of the immune suppressive growth factor TGF-β in mousecirculation. C57BL/6 mice were injected subcutaneously with B16F10melanoma cells (0.5×10⁶) and tumors were allowed to form. Mice were nextinjected 3× per week with 10 ug per mouse of cPG peptide. Serum wascollaged after 14 days. Serum was diluted in assay buffer and therelative levels of cytokines were examined by ELISA. FIG. 26A is a bargraph wherein data bars represent relative levels of TGF-0 as determinedby ELISA from control (DMSO) treated mouse and two different cPG peptidetreated mice. FIG. 26B is a bar graph wherein data bars representrelative levels of IL-4 as determined by ELISA from control (DMSO)treated mouse and two different cPG peptide treated mice. FIG. 26C is abar graph wherein data bars represent relative levels of IL-10 asdetermined by ELISA from control (DMSO) treated mouse and two differentcPG peptide treated mice.

FIG. 27A-FIG. 27B is a series of bar graphs showing that Mab HU177enhances the anti-tumor activity of the immune checkpoint inhibitoranti-PDL-1 antibody. Mice (C57BL/6) were injected with 3.5×10⁵ B16F10melanoma cells. Mice were allowed establish pre-existing tumors for 5days prior to treatment. Mice were treated (100 ug/mouse) with eitheranti-PD-L1 antibody alone, Anti-HU177 antibody alone or a combination ofboth antibodies 3 times a week for 15 days. FIG. 27A is a bar graphwherein data represents mean tumor volume at day 7+SE from 7 mice percondition. FIG. 27B is a bar graph wherein data represents mean tumorvolume at day 15+SE from 7 mice per condition.

FIG. 28 is a bar graph showing that synthetic collagen peptidescontaining the amino acid sequence, RGD, support T-cell adhesion.

FIG. 29 is a bar graph showing that monoclonal antibody, Mab XL313,inhibits interactions between human T-cell and the cryptic collagenepitope peptide P-2 (CQGPRGDKGEC; SEQ ID NO: 6).

FIG. 30 is a line graph showing that the integrin receptor αvβ3dose-dependently binds to the cryptic collagen epitope peptide P-2.

FIG. 31 is a bar graph showing that Mab XL313 inhibits recombinantintegrin receptor αvβ3 binding to the cryptic collagen epitope peptideP-2.

FIG. 32 is a photograph of an immunoblot showing that reducing theexpression of 3 integrin leads to reduced expression of PD-L1 in human Tcells.

FIG. 33 is a series of photographs of immunoblots showing that reducingthe expression of B3 integrin prevents stimulation of PD-L1 by the XL313cryptic collagen peptide P2 in human T cells.

FIG. 34 is a series of bar graphs showing that anti-XL313 antibodyreverses the inhibitory effects of the cryptic collagen peptide P2 onT-cell migration.

FIG. 35 is a series of photographs of immunoblots showing that thecryptic collagen peptide P2 induces expression of the immunosuppressivemolecule LAG3 in human T-cells.

FIG. 36 is a series of photomicrographs and a bar chart showing thatanti-XL313 Mab reduces the levels of the immune suppressive moleculeLAG-3 in melanoma tumors in vivo.

FIG. 37 is a bar chart showing that anti-XL313 Mab enhances the levelsof CD8+ T cells in melanoma tumors in vivo.

FIG. 38 is a bar chart showing that anti-HU177 Mab reduces the levels ofimmune suppressive CD4+/CTLA-4 positive T-Reg cells in melanoma tumorsin vivo.

DETAILED DESCRIPTION OF THE INVENTION

Described herein is an RGD containing cryptic collagen epitope that isgenerated in vivo. As described in detail below, rather than inhibitingαvβ3 signaling, this collagen epitope promoted αvβ3 activation andstimulated angiogenesis and inflammation. Additionally, an antagonist ofintegrin αvβ3 that inhibits signaling from this receptor was used toenhance the therapeutic activity of PD-1/PDL-1 targeting drugs for thetreatment of cancer and other diseases characterized by abnormal immuneresponse. Also described herein is an antagonist specifically directedto the HU177 cryptic collagen epitope that is used to enhance thetherapeutic activity of immune checkpoint targeting drugs for thetreatment of cancer.

Also described herein is an endothelial cell mechano-signaling pathwayin which a cryptic collagen epitope activates αvβ3 leading to a Src andP38MAPK-dependent cascade that leads to nuclear accumulation of YAP andstimulation of endothelial cell growth is defined. Collectively, thefindings provide evidence for a mechano-signaling pathway, but alsodefine a potential therapeutic strategy to control αvβ3 signaling bytargeting a pro-angiogenic and inflammatory ligand of αvβ3 rather thanthe receptor itself.

Angiogenesis, the process by which new blood vessels form frompre-existing vessels plays a critical role in normal and pathologicalevents. Efforts are underway to more precisely define the interconnectedmechanisms that control this crucial biological process in order todevelop more effective strategies to control neovascular diseases (1,2). Significant advances have been made in identifying molecularregulators of angiogenesis and their associated signaling pathways(3-5). A more precise understanding of angiogenic signaling pathways andthe networks of regulatory feedback loops operating within distinctcellular compartments has provided important clues to help explain themodest clinical impact of many anti-angiogenic strategies (1-5). Forexample, while vascular endothelial growth factor (VEGF) inducespro-angiogenic signaling leading to enhanced endothelial cell migration,growth, and survival, VEGF-induced blood vessels are often characterizedas immature, unstable, and leaky and can regress in the absence ofadditional signaling events (6). VEGF stimulation under specificcircumstances may lead to inhibition of angiogenesis in the context ofaltered PDGF signaling do to disruption of pericyte recruitment (6).These unexpected findings provide evidence of a negative role for theVEGF/VEGFR signaling during new vessel development (6). Similarly,studies have provided evidence for both a positive and a negative rolefor integrin αvβ3 in angiogenesis (7-9).

A wide array of alterations in the composition and biomechanicalproperties of extracellular matrix (ECM) proteins are known to occurduring angiogenesis and studies are beginning to define how thesechanges contribute to new blood vessel development (18, 21-23, 51-53).Among the key cell surface molecules that play roles inmechano-transduction to facilitate information flow from outside thecell to the inside are integrin receptors. Integrins may act likeinformation hubs by sensing diverse extracellular inputs and relayingthis information into a complex network of intracellular circuits thatultimately modulate cellular behavior (4). The precise molecularmechanisms by which cells fine-tune their response to changes within thestromal microenvironment are not completely understood. Furthercomplicating the understanding of new vessel development is theexpanding number of cell types that contribute to tissue specificcontrol of angiogenesis such as distinct subsets of stromal fibroblasts,progenitor cells and a variety of inflammatory cells such asneutrophils, mast cells and macrophages. The roles played by thesediverse cells during angiogenesis range from secretion of cytokines,chemokines and proteolytic enzymes to the differential expression ofother pro and anti-angiogenic factors. Thus, tight control mechanismsmust operate to allow coordination between these diverse compartments togovern tissue specific vascular responses.

Integrins are molecules with the ability to detect compositional andstructural changes within the ECM and integrate this information into anetwork of signaling circuits that coordinate context dependent cellbehavior. Among the most well studied integrins known to play a role inangiogenesis is αvβ3. The complexity by which αvβ3 regulatesangiogenesis is illustrated by the fact that this receptor may exhibitboth pro and anti-angiogenic functions (7-15). It is indicated that thedistinct biological responses stimulated by binding to αvβ3 may dependon many factors including the mechanical and biochemical features of theparticular ligands, the cell types within which αvβ3 is expressed aswell as the concentration and manner by which the ligands are presentedto the receptor (7-9,24-27). For example, studies indicate that αvβ3binding to specific NC1 domains of collagen or selected RGD peptides caninduce apoptosis, induce arteriole contraction and inhibit angiogenesis(19,29,54) while other αvβ3 ligands may promote cell survival, inducevascular dilation and support angiogenesis (21-23, 30, 55). Theseobservations are consistent with the notion that the final outcome ofαvβ3-mediated signaling may depend to a large extent on the particularcharacteristics of the ligand. While a wealth of data has shown that RGDpeptides can inhibit angiogenesis when administered exogenously, theapproach of using a cyclic RGD peptide to control tumor growth failed tosignificantly impact glioblastoma progression and patient survival inlate stage clinical testing (31). Interestingly, studies have indicatedthat specific RGD peptides may active β3 integrins (56, 57) and underdefined experimental conditions induce angiogenesis and tumor growth(30). These findings and other studies suggesting that amino acidsC-terminal to the RGD motif play roles in governing integrin selectivebinding, prompted the examination of the biological significance ofnaturally occurring RGD containing epitopes on angiogenesis.

For example, multiple pro-angiogenic roles have been proposed for αvβ3as cyclic arginine-glycineaspartic acid (RGD) containing peptides andantibodies targeting this integrin inhibit angiogenesis in animal models(10-12). In contrast, enhanced angiogenesis was detected in tumorsgrowing in αvβ3 null mice (13). Interestingly, reduced pathologicalangiogenesis was detected in transgenic mice expressing signalingdeficient 3 integrin, which resulted in part from defective recruitmentof bone marrow derived cells rather than specific endothelial celldefects (14). Moreover, evidence suggests that 3 integrin may play amore prominent role in the early stages of angiogenesis when new vesselsbegin to form, as reduction in endothelial cell expression of β3integrin impaired early stage pathological angiogenesis, but had littleeffect on later maturation stages once vessels had formed (15). Thesestudies, together with many others suggests αvβ3-mediated regulation ofangiogenesis is complex, temporally regulated and is not solelydependent on adhesive events, but also involves downstream signaling,the consequences of which may depend on the cell type and composition ofthe local extracellular microenvironment (4, 9, 12, 16). Because of theopposing biological responses observed following modulation of someangiogenic regulatory molecules, it is not surprising thatanti-angiogenic strategies based on targeting these factors have metwith limited clinical success.

Given the importance of integrin-extracellular matrix (ECM) interactionsin modulating the intensity and specificity of growth factor signaling(1-5, 17, 18), it is important to define how diverse components withinthe local vascular microenvironment function cooperatively to regulateangiogenesis. Interestingly, distinct αvβ3 ligands may stimulateopposing biological outcomes (7-15). For example, certain NC1 domains ofcollagen may bind αvβ3 and induce proapoptotic responses while bindingof other αvβ3 ligands may promote cell growth and survival (19-22).Given these findings and the complex biological effects observedfollowing direct targeting of αvβ3, an alternative therapeutic approachto control signaling from αvβ3 might involve specific targeting of thepro-angiogenic ligands of αvβ3 rather than directly targeting thereceptor itself. Proteolytic remodeling of the ECM can generate integrinbinding cryptic epitopes that play functional roles in angiogenesisincluding the LPG×PG containing HU177 cryptic epitope present inmultiple types of collagen and the HUIV26 cryptic epitope, which ispresent in collagen type-IV (21-23). While the HUIV26 epitope isrecognized by αvβ3, it is not specifically composed of an RGD motif(21).

Sequence analysis of RGD sites within collagen type-I indicate that theKGE tri-peptide motif that is C-terminal to the RGD site was highlyconserved among diverse species, while considerable variation isobserved in the other collagen RGD flanking sequences. While all five ofthe collagen RGD epitopes can support cell binding, the highly conservedRGDKGE (SEQ ID NO: 1) collagen peptide P-2 may play a functional roleangiogenesis and inflammation given that Mab XL313 directed to thisepitope but not an antibody that recognizes the other three RGD collagensites inhibited angiogenesis and inflammation in vivo. While the precisedifference between the three other naturally occurring non-RGDKGE (SEQID NO: 1) containing collagen epitopes, one or more of these epitopeswere detected in vivo in addition to the RGDKGE (SEQ ID NO: 1) epitope.Given that these RGD containing epitopes are thought to be largelycryptic and not readily accessible to cell surface receptor, thefindings are consistent with active collagen remodeling resulting ingeneration neoepitopes during new vessel formation.

Because of the importance of RGD sequences in mediating someintegrin-dependent interactions and the roles of amino acids flankingthe core RGD motif in establishing integrin-binding specificity andaffinity (24-27), the ability of RGD motifs within collagendifferentially regulate angiogenesis was determined. Sequence analysisof collagen type-I revealed that five different cryptic RGD motifs arepresent, each with unique flanking sequences. Surprisingly, theC-terminal KGE flanking sequence of one of these RGD motifs is highlyconserved in species as diverse as xenopous and man. In contrast,significant sequence and positional variation exists within the otherflanking sequences among different species.

ECM remodeling occurs as an early event during angiogenesis and multipleproteolytic enzymes including matrix metalloproteinase (MMPs) as well asserine and cysteine proteases the have the capacity to degrade intact orstructurally altered forms of collagen (18, 58). While the in vitrostudies indicate that MMP-2-mediated degradation of collagen can lead tothe generation of low molecular weights fragments recognized by MabXL313, the precise mechanism by which the RGDKGE (SEQ ID NO: 1) collagenepitope is generated in vivo is not completely understood. Analysis ofangiogenic CAM tissues suggests that a subset of macrophages may be animportant source of the RGDKGE (SEQ ID NO: 1) epitope. Activatedmacrophages with M2-like characteristics can express multiple enzymescapable of degrading collagen and in turn can internalize and furtherdegrade collagen into small low molecular weight fragments (35, 38).While little evidence exist that macrophages generate and deposit intacttriple helical collagen type-I, studies have indicated that certainisoforms of collagen may be expressed (59). Consistent with previousreports, intact collagen was detected; however low molecular weightRGDKGE (SEQ ID NO: 1) containing collagen fragments in both whole celllysates and serum free conditioned medium from macrophages like celllines was detected. While the studies do not rule out macrophagemediated collagen internalization as a contributing factor to the invivo generation the RGDKGE (SEQ ID NO: 1) collagen epitope, the in vitrostudies were carried out in the absence of serum or exogenously addedcollagen, and thus are consistent the active generation of the RGDKGE(SEQ ID NO: 1) collagen fragment by macrophages.

Activated macrophages including M2-polarized macrophages have beenimplicated in supporting angiogenesis and inflammation as multiplefactors secreted by these cells exhibit pro-angiogenic activities (35,60). While many studies indicate that synthetic RGD containing peptidesinhibit angiogenesis and tumor growth, the findings provide the firstevidence that macrophages may generate and release an RGDKGE (SEQ IDNO: 1) containing collagen epitope that may exhibit pro-angiogenicactivity. Importantly, previous studies have suggested that certainRGD-peptides can activate αvβ3 (56, 57) and may enhance vascularpermeability (45, 55), which might lead to release of inflammatoryfactors, which may in turn contribute to the formation of new bloodvessels.

To examine possible mechanisms by which the RGDKGE (SEQ ID NO: 1)collagen peptide might regulate angiogenesis endothelial cell receptorsfor this motif were identified. While the possibility that additionalnon-integrin receptors may bind this collagen epitope is not ruled out,the data suggest that αvβ3 can function as an endothelial cell receptorfor the RGDKGE (SEQ ID NO: 1) motif. Interestingly, αvβ3 bound both theRGDKGE (SEQ ID NO: 1) and RGDAPG (SEQ ID NO: 11) collagen peptides, yetonly RGDKGE (SEQ ID NO: 1) peptide significantly induced angiogenesisand inflammation in vivo. These findings are consistent with the notionthat distinct RGD containing αvβ3 ligands may promote differentbiological responses. Signaling downstream from αvβ3 is complex andstudies have indicated that simple binding of β3 integrin does notnecessarily lead to productive outside-in integrin signaling (61). Infact, the capacity of β3 integrins to promote outside-in signalingdepends on multiple factors including the extent of receptor clusteringand subsequent generation of mechanical tension within the actincytoskeleton, recruitment of adaptor and accessory proteins such asGal3, and Kindlin-2 and the association of the integrin with proteintyrosine phosphatases and certain growth factor receptors (62-65). Whilethe exact mechanisms leading to RGDKGE (SEQ ID NO: 1)-mediated αvβ3signaling is not completely understood, endothelial cell interactionswith the RGDKGE (SEQ ID NO: 1) peptide in the absence of serum led toenhanced phosphorylation of β3 integrin on tyrosine 747 and of Srcphosphorylation at tyrosine 416. These data and others are consistentwith an early mechanical mediated activation of β3 integrin that dependson Src given that blocking Src activity reduced β3 phosphorylationfollowing binding to the RGDKGE (SEQ ID NO: 1) motif.

Integrin signaling and Src activation are known to regulate thearchitecture of the actin cytoskeleton (66). Moreover, Src familykinases regulate P38MAPK, and activation of P38MAPK is thought toenhance actin stress fiber formation in endothelial cells and regulateangiogenesis in vivo (39-45). The findings provide insight into thecoordinated roles of P38MAPK and Src in regulating RGD-dependentendothelial cell signaling through αvβ3 as interactions with the RGDKGE(SEQ ID NO: 1) cryptic collagen epitope leads to enhanced P38MAPKphosphorylation in a Src-dependent manner. Moreover, RGDKGE (SEQ ID NO:1)-induced angiogenesis in vivo was associated with enhanced levels ofphosphorylated P38MAPK, and this angiogenic response was reduced by aninhibitor of P38MAPK. These findings are consistent with the notion thatRGDKGE (SEQ ID NO: 1) stimulated angiogenesis depends on P38MAPK.

Recent studies have suggested a role for actin stress fibers andmechanical tension in promoting nuclear accumulation of YAP, where it isthought to function in conjunction TEAD transcription factors inregulating gene expression (46-50). Given data suggesting a role for YAPin regulating endothelial cell growth and angiogenesis, the subcellulardistribution of YAP in endothelial cells following interaction with theRGDKGE (SEQ ID NO: 1) collagen peptide was examined. The data indicatethat endothelial cell interaction with the RGDKGE (SEQ ID NO: 1) epitopewas associated with enhanced nuclear accumulation of YAP. Integrinsignaling may play a role in the regulation of YAP as studies haveimplicated a role for β1 integrins expressed in skeletal stem cells andav integrins expressed in osteoblasts in governing YAP subcellularlocalization (67, 68). The findings are consistent with a mechanism bywhich αvβ3-mediated binding to the RGDKGE (SEQ ID NO: 1) epitope, butnot the related RGDAPG (SEQ ID NO: 11) epitope stimulates a signalingcascade leading to enhanced nuclear accumulation of YAP that depends onSrc and/or P38MAPK. This possibility is supported by the findings thatreduced levels of nuclear YAP was detected following αvβ3-mediatedinteraction with RGDKGE (SEQ ID NO: 1) peptide in endothelial cells inwhich Src or P38MAPK was inhibited. Given the documented role of YAP ingoverning cell growth coupled with the ability of the RGDKGE (SEQ IDNO: 1) collagen peptide to stimulate nuclear accumulation of YAP andenhance endothelial cell growth, it is possible that the RGDKGE (SEQ IDNO: 1) collagen peptide-induced endothelial cell growth is YAPdependent. Consistent with this possibility, no enhancement ofendothelial cell growth was detected following knockdown of YAP inendothelial cells stimulated with the RGDKGE (SEQ ID NO: 1) collagenpeptide, even though these cells are capable of proliferating asstimulation with VEGF or high levels of serum enhanced their growth.Given the studies, it is possible that part of the FGF-2 inducedangiogenic response observed in the chick CAM model might involve therecruitment of macrophages that generate a previously uncharacterizedRGDKGE (SEQ ID NO: 1) containing cryptic collagen epitope that binds toαvβ3 leading to Src and P38MAPK activation and nuclear accumulation ofYAP. Given that YAP is known to regulate a wide array of genes that mayimpact angiogenesis and inflammation including CTGF and Cry61, it islikely that the RGDKGE (SEQ ID NO: 1) collagen epitope may initiate acomplex pro-angiogenic program in vivo involving YAP-associatedregulation of multiple pro-angiogenic molecules and not simply berestricted to only enhancing endothelial cell growth.

Collectively, the results presented herein provide evidence that ahighly conserved RGDKGE (SEQ ID NO: 1) containing collagen epitope canbe generated by a subset of macrophages and the RGDKGE (SEQ ID NO: 1)collagen epitope can stimulate pro-inflammatory and angiogenic activity.Binding of the RGDKGE (SEQ ID NO: 1) collagen epitope to β3 integrin caninitiate a signaling pathway in endothelial cells leading to activationof Src and P38MAPK ultimately leading to nuclear accumulation of YAP andenhance cell growth. The results presented herein provide cellular andmolecular insight into how an endogenously generated RGD containingcryptic collagen epitope may promote rather that inhibit angiogenesis.Given the complexity of αvβ3 functions and the growing body of evidencethat the final outcome of αvβ3 binding may depend on the nature of theparticular ligand, the findings provide support for an alternativestrategy to help control the biological activity of β3 integrin byspecific targeting of endogenous pro-angiogenic ligands of αvβ3 ratherthan direct targeting of the receptor itself.

Integrin αvβ3 plays a functional role in promoting immune suppression inpart by upregulating the expression of the immune checkpoint regulatoryprotein PDL-1. Thus, targeting αvβ3 with function blocking (signalblocking) antagonists of αvβ3 or reducing expression of αvβ3 led toreduced expression of PDL-1. Thus antagonist of αvβ3 may enhance theanti-tumor efficacy of immune checkpoint therapy. Importantly, whileimmune check point inhibitors are known to provide some anti-tumoractivity in humans, this partial anti-tumor activity is only observed ina fraction of treated subjects. Described herein is the identificationof compounds and combination treatment strategies to enhance theefficacy of immune checkpoint inhibitors such as CTLA-4, PDL-1 and PD-1antibodies.

Therapeutic blockade of immune checkpoint regulatory molecules such asCTLA-4 and PD-1/PDL-1 signaling is known to be associated withsignificant immune related side effects including inflammation. Giventhese known side effects of immune checkpoint therapy and the ability ofspecific ligands of αvβ3 integrin such as the RGDKGE (SEQ ID NO: 1)containing collagen epitope to potentially induce inflammation in vivo(and the anti-stromal cell migratory activity of Mab HU177), combiningantagonist of αvβ3 (or an antagonist of HU177 or an antagonist of α10β1)with anti-PD-1/PDL-1 antagonists may reduce the inflammatory sideeffects associated with immune checkpoint inhibitor therapy.

Herein, is in vivo animal data, which indicate that melanoma tumors thatexpress integrin αvβ3 express the immune checkpoint protein PDL-1, whilethe same tumor cell type that was selected for lack of functional αvβ3exhibited little detectable PDL-1. Second, cellular interactions oftumor cells as well as endothelial cells with ECM proteins (denaturedcollagen and the RGDKGE (SEQ ID NO: 1) collagen epitope) that documentedligands of integrin αvβ3 lead to upregulated expression of PDL-1. Third,a function blocking antibody directed specifically to integrin αvβ3reduced expression of PDL-1 in tumor cells. Finally, an antibody (MabXL313) that specifically blocks the binding of an RGDKGE (SEQ ID NO: 1)epitope to αvβ3 and inhibits downstream signaling from αvβ3 enhanced theanti-tumor efficacy of an immune checkpoint inhibitor (anti-PDL-1) invivo.

Previous studies have indicated that blockade of immune checkpointproteins such as PDL-1, PD-1, and LAG-3 can result in some anti-tumoractivity. These anti-tumor effects, however were only partial and onlyoccurred in a fraction of the treated subjects. Thus, described hereinis the identification of compounds that enhance the effect of immunecheckpoint inhibitors such as antibodies targeting CTLA-4, PD-1 and/orPDL-1. To this end, studies have suggested that combining immunecheckpoint inhibitors with other chemotherapy drugs may enhance theanti-tumor activity. Importantly, a common side effect that can limitthe use of immune checkpoint inhibitors is the active induction ofinflammatory conditions such as dermatitis, pneumonitis and colitis.Part of the inflammatory process in vivo may involve alterations inexpression of inflammatory cytokines and infiltration of activatedstromal cells such as activated fibroblasts. In this regard, asdescribed in detail herein, antagonists of the XL313 epitope (Mab XL313)not only enhance the therapeutic activity of an anti-PDL-1 antibodytherapy in a mouse model (FIG. 9), but Mab XL313 epitope potentlyinhibits inflammation in vivo. Antagonists of the HU177 epitope (MabHU177) not only enhance the therapeutic activity of an anti-PDL-1antibody therapy in a mouse model (FIG. 27), but as described in detailbelow, Mab HU177 inhibits activated fibroblast migration andaccumulation within tumors in vivo. Additionally a αvβ3 binding RGDKGE(SEQ ID NO: 1) containing collagen epitope that stimulates αvβ3 integrinsignaling was shown to significantly enhance inflammation in vivo. Giventhese findings, it is possible that blocking αvβ3 signaling with anantagonist of αvβ3 would not only enhance the therapeutic activity of ananti-PDL-1/PD-1 or CTLA-4 based therapy, but may also potently inhibitinflammation in vivo. These findings are consistent with the notion thatcombining an antagonist of XL313 epitope (or antagonist of αvβ3) or anantagonist of HU177 epitope (or antagonist of α10β1) with immunecheckpoint inhibitor therapy not only enhances its efficacy, but alsoreduces the inflammatory side effects observed with the immunecheckpoint inhibitor therapy.

Described herein is evidence that an RGDKGE (SEQ ID NO: 1) containingcryptic collagen epitope is generated by a subset of macrophages andthis motif promoted rather than inhibited angiogenesis. These findingsare surprising given the wealth of experimental data indicating the highconcentration of RGD peptides inhibit rather than induce angiogenesis(11, 28, 29). Increasing evidence suggests that low concentrations ofcertain RGD peptides may actually enhance angiogenesis and tumor growth(30), which may explain at least in part the minimal impact of cyclicRGD peptide antagonists of αvβ3 and αvβ5 in human clinical trials (31).In addition to variations in concentrations that alter the biologicalresponse of certain RGD peptides, the specific composition of the aminoacids C-terminal to RGD motif within naturally occurring epitopes mayconfer unique pro-angiogenic and inflammatory activity. Taken together,these results are consistent with a mechanism by which the RGDKGE (SEQID NO: 1) collagen epitopes induce angiogenesis and inflammation bystimulating mechanical activation of αvβ3 leading to Src-dependentphosphorylation of P38MAPKinase that promotes nuclear accumulation ofthe Yes—associated protein (YAP) and enhanced endothelial cell growth.

EXAMPLES Example 1: Materials and Methods Reagents, Kits, Chemicals andAntibodies

Ethanol, methanol, acetone, bovine serum albumin (BSA), crystal violet,phosphate-buffered saline (PBS), purified human collagen type-IV andcollagen type-I, AMPA, 3,3,5,5′ tetramethybenzidine (TMB), phosphataseinhibitor cocktail, and cortisone acetate (CA) were from Sigma (StLouis, Mo.). MMP2 was from Chemicon/Millipore (Billerica, Mass.). FBSwas from Science Cell (Carlsbad, Calif.). Fibroblast growth factor-2(FGF-2) was obtained from R&D Systems (Minneapolis, Minn.).Nuclear/Cytoplasmic fractionation kit was from Thermo Scientific(Waltham, Mass.). P38MAPK inhibitor, SB202190 was obtained fromCalBoichem (San Diego, Calif.). RIPA buffer, protease inhibitor, and Srcinhibitor (PP2) were from Santa Cruz (Santa Cruz, Calif.). Anti-vWfantibody was from BD Pharmingen (San Diego, Calif.). Antibodies directedto P38MAPK, phospho-P38MAPK (Thr-180/Tyr-182), Src, and phospho-Src (Tyr416), were from Cell Signaling Technology (Danvers, Mass.). Antibodiesagainst tubulin, total binding protein (TBP), YAP, β3, and phospho-133(Tyr747) were from Santa Cruz (Santa Cruz, Calif.). Anti-Igfbp4 andanti-MMP9 antibodies were obtained from Abcam (Cambridge, Mass.).Function blocking antibodies P4C10 (anti-β1), LM609 (anti-αv β3) andP1F6 (anti-αv β5) were from R&D Systems (Minneapolis, Minn.).HRP-conjugated secondary antibodies were from Promega (Madison, Wis.).Anti-collagen type-I antibody was from Rockland (Limerick, P A) andanti-collagen type-IV was from Millipore (Billerica, Mass.). Mousemonoclonal antibodies XL313, and XL166 were developed. Alexa-488,Alexa-594, streptavidin Alexa-594, and phalloidin Alexa-594 labeledantibodies were from Invitrogen (Carlsbad, Calif.). Synthetic collagenRGD containing peptides (P-1; CKGDRGDAPGC (SEQ ID NO: 5), P-2;CQGPRGDKGEC (SEQ ID NO: 6), P-3; CAGSRGDGGPC (SEQ ID NO: 7), P-4;CQGIRGDKGE (SEQ ID NO: 8), P-5; CRGPRGDQGPC (SEQ ID NO: 9) and peptidecontrol (P-C; CQGPSGAPGEC; SEQ ID NO: 10) were obtained from QEDBiosciences (San Diego, Calif.).

Cells and Cell Culture

RAW 264.7 and THP-1 cells were from ATCC (Manassas, Va.) and cultured inDMEM and RPMI respectively in the presence of 10% FBS, 1.0% pen-strepand 1.0% sodium pyruvate. Immortalized BV-2 cells, and were cultured inDMEM with 10% FBS, 1.0% pen-strep and 1.0% sodium pyruvate. Human dermalfibroblasts (HDF) were obtained from Science Cell (Carlsbad, Calif.) andcultured in fibroblast growth medium with 2.0% FBS and used betweenpassages 4 to 9. Human retinal microvascular endothelial cells (HRMVECs)were obtained from Applied Cell Biology Institute (Kirkland, Wash.) andcultured in EBM2 supplemented with EGM-2 growth factors. Human umbilicalvein endothelial cells (HUVECS), human microvascular endothelial cells(HMVECS) were obtained from ATCC (Manassas, Va.) and cultured in EBM2with supplemental growth factors EGM-2 or EGM-2MV respectively. Allendothelial cell growth media contained 2% FBS, 1.0% pen-strep and 1.0%sodium pyruvate and used experimentally between passages 3 to 9. Forcollection of conditioned media, cells were cultured in basal mediaunder serum free conditions for 24 hours. Conditioned media wascollected and concentrated 10× using an Amicon Ultracell, 3 kDacentrifugal ultrafiltration cartridge.

Cell Adhesion Assays

RGD peptides (P-1, P-2, P-3, P-4, P-5, and P-C) were immobilized (100μg/ml) to wells. HUVEC, HMVEC, HRMVEC, and HDF cells were suspended inadhesion buffer (RPMI containing 1 mM MgCl2, 0.2 mM MnCl2 and 0.5% BSA)and 1×105 cells were seeded into the wells in the presence or absence ofP4C10, LM609, P1F6 or control antibodies (100 μg/ml). Cells were allowedto attach for 25 min at 37° C. Media containing non-attached cells wasaspirated and attached cells were washed with PBS and stained withcrystal violet. Cell adhesion was quantified by measuring the opticaldensity of eluted dye. Adhesion assays were completed at least threetimes with triplicate wells.

Collagen Proteolysis and Solid Phase Binding Assays

Collagen type-I and collagen type-IV were heat denatured for 15 min andthen incubated with APMA-activated MMP2 for 0.5 h, 1 h, 4 h, 8 h, and 20h, at 37° C., followed by a five minute boil to deactivate remainingMMP. For solid phase ELISAs, synthetic RGD-containing collagen peptides(P-1, P-2, P-3, P-4, P-5, and P-C) were immobilized (100 μg/ml) to wellsor wells were, coated with 10 μg/ml of native or MMP2-proteolyzedcollagen type-I or type-IV. Wells were blocked with 1% BSA in PBS for 1hour and then incubated with 1 μg/ml of Mabs XL313 or XL166 for 1 h,washed, and incubated with antimouse HRP-conjugated antibodies (1:5000).Bound Mabs were detected with a TMB substrate as per manufacturesinstructions and quantified via spectrometer measurements. All assayswere carried out at least four times in triplicate wells.

Chick Cam Inflammation and Angiogenesis Assays

The chick chorioallantoic membrane (CAM) assays were carried out withsome modifications (19). For all experiments CAMs of 10-day-old chickembryos obtained from Charles River (North Franklin, Conn.) wereseparated from the shell membrane. Filter discs either non-treated(inflammation assays) pretreated (angiogenesis assays) with cortisoneacetate (3.0 mg/ml) containing RPMI only or FGF-2 (40 ng). CAMs wereeither non-treated or treated topically with Mab XL313, XL166 or anon-specific control antibody (10 μg/embryo every 24 h for threeconsecutive treatments). For peptide induction experiments, CAMs werestimulated with 100 ng/ml of the RGD peptides (P-1, P-2, P-C) in thepresence or absence of the P38MAPK inhibitor, SB202190 (10 μM). At theend of the incubation period the embryos were sacrificed and the CAMtissues were analyzed. Angiogenesis was quantified by counting thenumber of angiogenic branching blood vessels within the area of thefilter disc. The angiogenic index was determined by subtracting the meannumber of blood vessel branch points from untreated CAMs from eachexperimental condition. Eight to twelve embryos were used per conditionand experiments were repeated at least three times.

Immunohistochemistry and Immunofluorescence Analysis

CAMs examined for accumulation of granulation tissue were harvested,fixed in 4% PFA and paraffin embedded and sectioned (4 μm) and stainedwith Giemsa. CAM tissues analyzed via immunofluorescence were harvested,embedded with OCT, snap frozen and sectioned (4 μM). Frozen sectionswere fixed in 50% methanol/50% acetone, air dried and blocked with 2.5%BSA for 1 h at 37° C. For expression of the XL313 and XL166 epitopes aswell as monocyte/macrophages, FGF-2 stimulated CAM tissues were stainedwith Mabs XL313, XL166 (50 μg/ml) or KUL1 (1:250) and then incubatedwith Alexa-488 labeled secondary antibodies (1:2000 dilution).Co-staining of monocytes/macrophages and the Mab XL313 reactive epitopein FGF-2 stimulated CAMs was performed by sequential staining by firstprobing with KUL1 (1:250) and then with a biotinconjugated Mab XL313 (50μg/ml), and with secondary antibodies Alexa-488 and streptavidinAlexa-594 (1:2000) respectively. Using a similar strategy, sections ofRGD peptide-treated CAM tissues were analyzed for phosphorylated-P38MAPKin angiogenic vessels by co-incubation of anti-phospho-P38MAPK (1:50)and anti-vWf (1:500) antibodies. Subcellular localization of YAP wasobserved in HUVECS attached to P-2 and P-1 coated glass coverslips.Cells were allowed to attach for 15, 30, or 60 min in the absence ofserum and were fixed with 4% PFA. Fixed cells were washed and blockedwith 2.5% BSA and stained with anti-YAP (1:200) and phalloidin (1:500).All sections and slides were counter stained with DAPI.

Cell Proliferation Assays

HUVECs (WT, shYAP1 or control transfected) or HRMVECs were plated at2,000 cells per well with complete EGM-2 media containing 2.0% FBS inthe absence or presence of P-1, P-2 or P-C (100 ng/ml) and allowed togrow for 24 hours. Cell growth was monitored using a BrdU or MMT assaykits according to manufacturer's instructions. All assays were completedat least three times in triplicate wells.

Inhibitor Experiments

Endothelial cells (HUVECs or HRMVECs) were incubated in serum free mediawith 1 mM MgCl₂, 0.2 mM MnCl2 in the presence of a Src inhibitor, PP2,(10 μM) a P38MAPK inhibitor, SB202190 (10 μM) or vehicle only (DMSO) for10 min at 37° C. Treated cells were then seeded on to immobilized P-1and P-2 and lysates were collected at 15 min.

Western Blots

Whole cell and CAM tissue lysates were collected in RIPA buffersupplemented with 1× protease inhibitor and 1× phosphatase inhibitor andwere run on polyacrylamide gels using denaturing conditions. Prior toloading the gels, 6× sample buffer was added to each of the lysates(final concentration 1×) and boiled for five minutes. Twenty to 50 μg oftotal protein were loaded into each lane. For detection of proteinslarger than 50 kD, 10% gels were used; for proteins smaller than 50 kD15% gels were used. Gels were run at 60 volts (v) until the dye frontpassed through the stacking gel, and then the voltage was increased to100V for the remainder of the separation. Precision Plus proteinstandards (Bio-Rad) were used to visualize migration. Protein wastransferred to nitrocellulose membranes using a wet tank system andblocked for 1 h using 10% non-fat dried milk in tris-buffered salinewith 0.01% Tween-20 (TBS-T). Membranes were incubated with primaryantibodies (anti-coll-I (1:250), anti-coll-IV (1:250), Mab XL313 (2μg/ml), Mab XL166 (2 μg/ml) anti-β-actin (1:5000), anti-phospho-β3(1:7000), anti-β3 (1:1000), anti-phospho-Src (1:500), anti-Src (1:500),anti-phospho-P38MAPK (1:500), anti P38MAPK (1:2000), anti-YAP (1:500),anti-Tubulin (1:2000), anti-TBP (1:1000)) in 5% BSA in TBS-T overnightat 4° C. with gentle agitation. Membranes were washed 3 times in TBS-Tfor five minutes. Blots were then incubated with HRP conjugatedsecondary antibodies (1:15000) in 1% non-fat milk in TBS-T for 1 h.Membranes were washed a second time as indicated above and exposed tochemiluminecent substrate for three minutes prior to exposure toautoradiography film in a dark room. Western blot bands were quantifiedusing Image J software based on pixel intensity.

Transfections and Lentiviral Transductions

Raw cells were transfected with 1 μg of HuSH shRNA plasmids for collagentype I, alpha 2 using Amaxa cell line nucleofector kit V (program#T024). Constructs expressing 21-nucleotide short hairpin RNAs (shRNA)targeting human YAP (shYAP) or non-targeting control (shNT,Sigma-Aldrich, SHC002) were used. Humantargeting shYAP1 lentiviral shRNAwas obtained from the Thermo Scientific RNAi consortium(TRCN0000107625). Constructs were packaged into lentivirus, pseudotypedwith the vesicular stomatitis virus glycoprotein. Transduction wasperformed by incubating cells with lentivirus, and stably transducedcells were subsequently used for studies. All cell lines were verifiedby morphology and mouse and human YAP-specific PCR. The efficacy of YAPknock down was determined to be between 70%-80%. Endothelial cells werecertified mycoplasma-negative by PCR (Lonza), and primary cell cultureswere used within the indicated passage numbers. Cells were transducedand selected using puromycin.

Statistical Analysis

Statistical analysis was performed using the InStat statistical programfor Macintosh computers. Data were analyzed for statistical significanceusing Student T test. P values <0.05 were considered significant.

Example 2: Cryptic RGD Containing Peptides from Collagen Type-1 SupportCell Adhesion

Studies have documented the capacity of extracellular matrix (ECM)proteins containing the short amino acid sequence RGD to supportinteractions mediated by integrin receptors (33). The ability of cellsto interact with RGD sites within the context of larger glycoproteinsdepends on many factors, some of which include the adjacent flankingsequences surrounding the core RGD tri-peptide as well as thegeometrical configuration of the intact molecule and how these moleculesare oriented within the context of the interconnected network of otherECM proteins (24, 25, 33). Flanking sequences immediately C-terminal tothe RGD site can govern integrin selective binding (24, 25, 33). RGDmotifs can be cryptic and inaccessible to cell surface receptors as isillustrated in the case of triple helical collagen (34). In this regard,five different cryptic RGD containing sites exist within human collagentype-I, each with distinct flanking sequences (Table 1).

TABLE 1 RGD containing epitopes of collagen type-I. PeptideAA Sequence (SEQ ID NO:) Location P-1 KGDRGDAPG (SEQ ID NO: 2) Colla1742-750 P-2 QGPRGDKGE (SEQ ID NO: 3) Colla1 1090-1098 P-3AGSRGDGGP (SEQ ID NO: 12) Colla2 774-782 P-4 QG1RGDKGE (SEQ ID NO: 13)Colla2 1002-1010 P-5 RGPRGDQGP (SEQ ID NO: 14) Colla2 819-827 P-CQGPSGAPGE (SEQ ID NO: 15) NA

Five different cryptic RGD containing sites exist within human collagentype-I, each with distinct flanking sequences. Synthetic peptides ofthese five sequences were generated and designated P-1 through P-5 asshown above. Additionally, a control peptide (P-C) was generated lackingthe RGD tri-peptide motif. The sequences in Table 1 correspond to thefollowing SEQ ID NOs.: KGDRGDAPG (SEQ ID NO: 2), QGPRGDKGE (SEQ ID NO:3), AGSRGDGGP (SEQ ID NO: 12), QGIRGDKGE (SEQ ID NO 13); RGPRGDQGP (SEQID NO: 14); and QGPSGAPGE (SEQ ID NO: 15).

RGD Peptides are Capable Supporting Cell Adhesion

Due to the importance of the RGD tri-peptide motif, the flankingsequences within collagen type-I that surround the core RGD site mayalter its cellular recognition were determined. To assess whether thesecryptic collagen RGD motifs were redundant or whether the flankingsequences help convey distinct properties, each of the collagen type-IRGD epitopes were synthesized, along with their associated flankingsequences. The five different RGD peptides were immobilized and theirability to facilitate cell adhesion was examined. As shown in FIG. 1A,all five collagen RGD peptides (P1-P5) support human umbilical veinendothelial cell (HUVEC) adhesion, with peptide 5 (P-5) promoting thehighest levels of adhesion. In contrast, a peptide control (P-C) inwhich the RGD motif of P-2 was replaced with SGA failed to supportadhesion. To confirm that the adhesion promoting ability was notspecific to only HUVECs similar assays were carried out with humanmicrovascular endothelial cells (HMVEC), human retinal microvascularendothelial cells (HRMVEC) and human dermal fibroblasts. All endothelialcells and fibroblast bound the five RGD containing collagen peptideswhile the control peptide failed to support interactions (FIG. 1B-FIG.1D). These data indicate that while some differences were observed, allfive RGD peptides were capable supporting cell adhesion.

Cryptic Collagen RGD Epitopes Using Monoclonal Antibodies

To study these cryptic collagen RGD epitopes, monoclonal antibodies weregenerated. Two distinct antibodies were isolated with the ability tospecifically discriminate between different RGD containing epitopes. Asshown in FIG. 1E, Mab XL313 specifically bound to collagen peptides P-2and P-4 containing the conserved RGDKGE (SEQ ID NO: 1) motif (Table 1),but showed no significant reactivity with other RGD peptides includingP-1, P-3 or P-5. A second antibody termed XL166 recognized RGDcontaining collagen peptides P-1, P-3 and P-5, but failed to bind to theRGDKGE (SEQ ID NO: 1) containing peptides P-2 and P-4 (FIG. 1F). NeitherMab XL313 nor XL166 showed interaction with the control peptide (P-C).

Example 3: XL313 Exhibits Selective Binding for Proteolyzed CollagenType-I

The capacity of Mab XL313 to bind its RGD motif within the context ofthe full-length collagen molecule was determined. To facilitate thesestudies, denatured collagen type-1 or IV was incubated with MMP-2 for 12hrs to generate proteolyzed collagen. MMP-2 mediated proteolysis ofcollagen type-I and IV resulted in the generation of multiple fragmentsas indicated by Western blot analysis using antibodies specificallydirected to either collagen type-I (FIG. 2A left) or collagen type-IV(FIG. 2A right). Mab XL313 specifically directed to the RGDKGE (SEQ IDNO: 1) collagen sequence exhibited minimal reactivity with intactcollagen type I or type IV under denaturing and reducing conditions(FIG. 2B) or under non-denaturing and non-reducing conditions of theELISA (FIG. 2C). In contrast, Mab XL313 readily detected low molecularweight fragments of collagen type-I, but not collagen type-IV followingproteolysis (FIG. 2B). Mab XL313 failed to recognize other RGDcontaining ECM proteins including vitronectin or fibronectin.

To further examine the generation of the low molecular weight RGDKGE(SEQ ID NO: 1) containing collagen fragments, a time course of MMP-2mediated collagen proteolysis was examined. MMP-2 mediated degradationof collagen type-I resulted in a time dependent generation of Mab XL313reactive collagen fragments (FIG. 2D). The majority of the collagen wasproteolyzed into Mab XL313 reactive fragments of approximately 14 Kd to16 Kd by 8 hours. These findings indicated that XL313 collagen epitopeswere cryptic within the intact non-denatured collagen type-1 moleculeand that proteolytic degradation was required to efficiently expose thehidden RGDKGE (SEQ ID NO: 1) containing motif.

Example 4: Differential Roles of Cryptic RGD Collagen Motifs onAngiogenesis and Inflammation In Vivo

Studies have shown that collagen remodeling within the vascular basementmembrane can result in exposure of multiple non-RGD cryptic collagensites including the HUIV26 and HU177 epitopes that can play active rolesin angiogenesis (21, 23). Given these findings, it was determinedwhether distinct RGD containing epitopes were exposed in vivo. First, toexamine whether these RGD epitopes could be generated duringangiogenesis the chick chorioallantoic membrane (CAM) model (19) wasused. The CAMs of chick embryos were stimulated with FGF-2, and thegeneration of RGD containing collagen epitopes was examined using MabsXL313 and XL166. As shown in FIG. 3A, little Mab XL313 or XL166 reactiveepitope was detected in non-stimulated CAMs. In contrast, RGD containingcollagen epitopes (Green) were readily detected in FGF-2 stimulated CAMtissues confirming the differential generation of RGD containingepitopes recognized by these antibodies. Mab XL313 and XL166 reactiveRGD epitopes did not exhibit a typical extracellular fibril collagenpattern, but rather exhibited punctate distribution with bothintracellular and scattered extracellular localization. Theintracellular distribution is similar to the staining pattern ofcollagen degradation products previously documented within intracellularvesicles of macrophages (35).

Given the generation of these distinct sets of RGD containing epitopes,their active roles in regulating angiogenesis and inflammation wereexamined. Angiogenesis was induced within the CAMs of 10-day old chickembryos with FGF-2 using filter discs coated with cortisone acetate (CA)to reduce growth factor associated inflammation. As shown in FIG. 3B,treatment with Mab XL313 directed to the cryptic RGDKGE (SEQ ID NO: 1)containing epitope (P<0.05) inhibited FGF-2 induced angiogenesis bygreater than 75% as compared to non-treated or control antibody. MabXL166 that specifically binds the remaining three cryptic RGD collagenpeptides (P-1, P-3, and P-5) had no effect. These findings wereconsistent with the notion that while distinct RGD containing collagenepitopes were readily generated in vivo, were not functionallyredundant.

FGG-2 Induced Inflammation is Associated with Recruitment andAccumulation of Granulation-Tissue Associated Macrophages.

The chick CAM has been routinely used to assess inflammation andgranulation tissue formation, which are largely dependent oninfiltration of chick heterophils (the avian equivalent of neutrophils),macrophages and activated fibroblasts (36, 37). To study the potentialdifferential biological impact of RGD epitopes, FGF-2 inducedinflammation was examined by carrying out similar experiments in theabsence of cortisone acetate and quantifying CAM thickening. As shown inFIG. 3C, treatment with FGF-2 induced CAM inflammation as indicated byrobust tissue thickening (top panels), extensive infiltration ofinflammatory infiltrates as indicated by Giemsa stain (middle panel) andincreased accumulation of monocytes and macrophages (bottom panel)following staining with an antibody directed to avian specific monocytesand macrophages. Macrophage infiltration of the CAMs stimulated withFGF-2 was (P<0.05) enhanced over 2-fold as compared to control (FIG. 3D)indicating that FGF-2 induced inflammation in this model is associatedwith the recruitment and accumulation of granulation-tissue associatedmacrophages.

FGF-2 Induced Inflammatory Response in the Presence or Absence ofAnti-RGD Specific Antibodies of XL313 and XL166

To examine whether the RGD containing collagen epitopes play a role inthe FGF-2 stimulated inflammatory response, this FGF-2 inducedinflammatory response was examined in the presence or absence ofanti-RGD specific antibodies XL313 and XL166. Quantification indicatedthat FGF-2 stimulation in the absence of cortisone acetate resulted inapproximately 50% of the CAMs showing robust formation of thickgranulation tissue (FIG. 3E). Treatment of CAMs with cortisone acetate,a well document anti-inflammatory agent significantly (P<0.05) reducedthe percentage of CAMs exhibiting thickening by greater than 80% ascompared to either no treatment or control antibody. Interestingly,treatment of CAMs with Mab XL313 also (P<0.05) inhibited the number ofCAMs exhibiting inflammation by approximately 75% as compared tocontrols. In contrast, treatment of CAMs with Mab XL166 showed nosignificant (P>0.05) impact. These data are consistent findings whileexamining angiogenesis indicating a differential role for specificcryptic RGD epitopes in vivo.

Example 5: Generation of XL313 Cryptic RGDKGE (SEQ ID NO: 1) Epitope

During angiogenesis and inflammation, multiple cell types includingendothelial cells, fibroblasts and macrophages proteolytically remodelextracellular collagen creating a permissive microenvironment thatfacilitates stromal cell infiltration and new blood vessel growth. Avariety of cells including fibroblasts and endothelial cells expresscollagen and, partially degraded collagen can be internalized andfurther processed by activated M2-like macrophages leading to thegeneration of low molecular weight fragments (35, 38). Because of theunique pattern of Mab XL313 immunoreactivity observed in vivo, it wasdetermined whether stromal cells associated with angiogenesis andinflammation could generate the RGDKGE (SEQ ID NO: 1) containingepitope. Whole cell lysates were prepared from fibroblasts andendothelial cells and Western blots were performed. While some Mab XL313reactive species was detected in lysates of endothelial cells andfibroblasts, the major immunoreactive species migrated betweenapproximately 75 Kd to 28 Kd indicating that these species were unlikelyto represent intact collagen. In addition, minimal amounts of lowmolecular weight species were detected that corresponds to the major 14Kd to 16 Kd collagen fragments detected following MMP-2 mediatedproteolysis. While a small amount of an approximately 20 Kd XL313reactive species was detected in serum free conditioned medium (CM)collected from HUVECS, no significant levels of immunoreactive fragmentsin fibroblast CM was detected.

Given the minimal reactivity observed in these cell types known toexpress collagen, and that FGF-2 induced a strong angiogenic andinflammatory response in CAMs that was associated with an extensiveinfiltration of macrophages, FGF-2 treated CAM tissues for theco-distribution of the XL313 epitope and macrophages was examined. Asshown in FIG. 4B, the XL313 reactive epitope (Red) co-localized with asubset of macrophages (Green) in FGF-2 stimulated CAMs, indicating thatmacrophages may be one source of the XL313 collagen epitope. To studythis possibility, both whole cell lysates (FIG. 4C left) and serum freeCM (FIG. 4C right) from three different macrophage-like cell lines wereexamined. In contrast, to endothelial cells and fibroblasts, strongimmunoreactive species were readily detected in cell lysates and CM frommultiple macrophage cell lines including Raw 264.7, THP1 and BV2.Importantly, low molecular weight immunoreactive species migratingbetween 14 Kd to 16 Kd were readily detected in serum free CM (FIG. 4Cright). These 14 Kd to 16 Kd immunoreactive species corresponded to thesize of low molecular weight Mab XL313 reactive collagen fragmentsdetected following MMP-mediated proteolytic digestion of purifiedcollagen. To further study the XL313 immunoreactive species detected inmacrophages, the generation of the 14 Kd to 16 Kd RGDKGE (SEQ ID NO: 1)containing XL313 fragments in Raw 264.7 macrophages was examined. First,to examine the expression of collagen in these macrophages, theexpression of mRNA for the alpha 1 and alpha 2 chains of collagen type-Iin RAW macrophages by PCR was confirmed. Secondly, while no full lengthcollagen type-I was detected in either cell lysates or CM frommacrophages using anti-collagen type-I specific antibodies, lowmolecular weight fragments migrating at the same molecular weight as theRGDKGE (SEQ ID NO: 1) containing epitope in both cell lysates and CMusing either Mab XL313 or an anti-collagen-I specific antibody (FIG. 4D)was detected. Importantly, shRNA-mediated knockdown of the alpha 2 chainof collagen type-I reduced immune-detection of the low molecular species(14 Kd to 16 Kd) in both lysates and CM when using either anti-collagenspecific antibodies or Mab XL313 (FIG. 4D). Together, these findingswere consistent with the possibility that macrophages were one cellularsource of the RGDKGE (SEQ ID NO: 1) collagen epitope.

Example 6: Induction of Angiogenesis and Inflammation In Vivo by SolubleRGDKGE (SEQ ID NO: 1) but not a Related RGDAPG (SEQ ID NO: 11)Containing Collagen Epitope

Due to the differential expression and bio-distribution of the RGDKGE(SEQ ID NO: 1) epitope in angiogenic CAM tissues and its expression inmacrophage-conditioned medium, it was determined whether a solublecirculating form of this RGDKGE (SEQ ID NO: 1) epitope could begenerated. To examine this possibility, chick embryos were eitherun-treated or stimulated with FGF-2 and serum, and collected three dayslater. As shown in FIG. 5A, low levels of circulating Mab XL313immunoreactive epitope was detected in the serum from non-stimulatedcontrol chick embryos. In contrast, a (P<0.05) 2-fold increase wasdetected in the levels of circulating XL313 epitope following FGF-2stimulation. These findings indicated the differential release of asoluble form of this RGDKGE (SEQ ID NO: 1) containing collagen epitope.

Soluble Peptide Containing the XL313 Epitope Actively RegulatesAngiogenesis and Inflammation

Because a soluble form of the RGDKGE (SEQ ID NO: 1) epitope was detectedin vivo, it was next determined whether a soluble peptide containing theXL313 epitope actively regulated angiogenesis and inflammation. Toexamine this possibility, the effects of the RGDKGE (SEQ ID NO: 1)containing collagen peptide on angiogenesis and inflammation in thechick CAM were assessed. As shown in FIG. 5B, the XL313 RGDKGE (SEQ IDNO: 1) containing collagen peptide (P-2) dose dependently enhancedangiogenesis with maximum induction observed at a dose of 100 ng/CAM.Stimulation of CAMs with the RGDKGE (SEQ ID NO: 1) containing collagenpeptide P-2, but not the related RGDAPG (SEQ ID NO: 11) containingcollagen peptide P-1, or peptide control (P-C) significantly (P>0.05)induced angiogenesis as compared to no treatment (FIG. 5C).

The effects of the soluble RGD containing collagen peptides oninflammation were examined. As shown in FIG. 5D, FGF-2 induced a stronginflammatory response in the chick CAM in the absence of cortisoneacetate as indicated by approximately 55%-60% of the CAMs exhibitingextensive tissue thickening. Minimal evidence of tissue inflammation wasobserved following stimulation with either control peptide (P-C) or theRGDAPG (SEQ ID NO: 11) containing collagen peptide P-1, while the RGDKGE(SEQ ID NO: 1) collagen peptide P-2 significantly (P<0.05) inducedinflammation to nearly that of FGF-2 stimulation (FIG. 5D). Thesestudies indicated that the biological impact of RGD containing collagenpeptides on angiogenesis and inflammation in the chick CAM depended ontheir associated flanking sequences.

Example 7: RGDKGE (SEQ ID NO: 1) Containing Peptide P-2 InducedAngiogenesis Depends on P38MAPK

Studies have indicated that angiogenesis and inflammation in the chickCAM is associated with alterations in MAP kinase signaling includingP38MAPK (40, 41). To examine mechanisms that regulate angiogenesisfollowing stimulation with the RGDKGE (SEQ ID NO: 1) containing collagenpeptide P-2, CAM tissues from untreated or RGD peptide treated animalswas examined. As shown in FIG. 6A, elevated levels of phosphorylatedP38MAPK were detected in lysates from CAMs treated with RGDKGE (SEQ IDNO: 1) containing peptide P-2 as compared to either untreated or RGDAPG(SEQ ID NO: 11) containing peptide P-1 treated animals. Quantificationof CAM tissues (N=12) indicated a P<0.05) 4-fold increase inphosphorylation of P38MAPK as compared to controls (FIG. 6B). Consistentwith studies, while activated P38MAPK was detected in multiple celltypes in chick CAMs, co-staining analysis indicated expression ofphosphorylated P38MAPK in vWf positive blood vessels (FIG. 6C).

The effects of the RGD containing collagen peptides on the levels ofphosphorylated P38MAPK in endothelial cells were examined. As shown inFIG. 6D and FIG. 6E, while both RGD containing collagen peptides P-1 andP-2 support cell binding, interactions with RGDKGE (SEQ ID NO: 1)collagen peptide P-2 resulted in enhanced phosphorylation of P38MAPK ascompared to RGDAPG (SEQ ID NO: 11) collagen peptide P-1 in HUVECs (FIG.6D) or HRMVEC (FIG. 6E). A functional role for P38MAPK in mediatingRGDKGE (SEQ ID NO: 1) peptide P-2 induced angiogenesis in vivo wasdemonstrated as P-2 induced angiogenesis was (P<0.05) reduced by P38MAPKinhibitor (FIG. 6F). Together, these data indicate a role for P38MAPK inthe peptide P-2 stimulated pro-angiogenic response observed in vivo.

Example 8: Collagen Peptide P-2 Binds and Activates αvβ3 and StimulatesP38MAPK Activation in a Src Dependent Manner

It is well established that multiple integrins recognize RGD amino acidmotifs within ECM proteins. However, the ability of an integrin to binddistinct RGD epitopes depends in part on its orientation within theparent molecule and the C-terminal amino acid sequences flanking the RGDmotif (25-27). Potential cell surface receptors that mediateinteractions with the RGDKGE (SEQ ID NO: 1) containing collagen P-2peptide were identified. The ability of endothelial cells to bind theRGD collagen peptides P-1 and P-2 in the presence or absence of functionblocking anti-integrin antibodies was examined. As shown in FIG. 7A,anti-β1 specific antibody had minimal effect on HUVEC adhesion topeptide P-2 while anti-αvβ3 antibody (P<0.05) inhibited interactions byapproximately 90% as compared to control. Anti-αvβ5 antibody showedlittle inhibition. Similar results were observed with P-1, whichcontains the RGDAPG (SEQ ID NO: 11) sequence (FIG. 7B). These dataindicate that αvβ3 functions as a receptor for both RGD containingpeptides in endothelial cells.

RGD Peptides Differentially Altered αvβ3-Mediated Signaling

Because both RGD containing peptides bind αvβ3 coupled with thedifferential effect of these peptides on angiogenesis and inflammation,it was determined whether the RGD peptides differentially alteredαvβ3-mediated signaling. Endothelial cells were allowed to attach to P-1and P-2 and the relative level of β3-integrin phosphorylation wasexamined. As shown in FIG. 7C, in the absence of any growth factors, β3phosphorylation (P<0.05) increased over 3-fold following ligation ofRGDKGE (SEQ ID NO: 1) peptide P-2, as compared to RGDAPG (SEQ ID NO: 11)peptide P-1. Ligand binding of 3 integrin induced receptor clusteringand initiated down-stream signaling events including activation ofprotein kinases including Fak and Src. Therefore, the differentialphosphorylation of these protein kinases following the earlymechanical-mediated interactions with the RGD containing collagenpeptides was assessed. Minimal Fak phosphorylation was detectedfollowing ligation of either P-1 or P-2 under these serum freeconditions. In contrast, Src phosphorylation was enhanced by nearly2-fold following interactions with P-2 as compared to P-1 (FIG. 7D).Moreover, activation of 3 integrin and P38MAPK in endothelial cellsfollowing binding to P-2 was dependent on Src as incubation ofendothelial cells with a Src inhibitor reduced β3 and P38MAPKphosphorylation following binding to collagen peptide P-2 (FIG. 7E andFIG. 7F). Activation of Src and P38MAPK leads to enhanced actinpolymerization and stress fiber formation (43-45), therefore the actincytoskeleton in endothelial cells following binding to the RGD collagenpeptides was examined. Endothelial cell interactions with the RGDKGE(SEQ ID NO: 1) collagen peptide P-2 resulted in accelerated actin stressfiber formation as compared to interactions with RGDAPG (SEQ ID NO: 11)collage peptide P-1 (FIG. 7G). Cumulatively, these findings indicate theability of the conserved RGDKGE (SEQ ID NO: 1) collagen epitope tostimulate initiation of a β3-integrin dependent mechano-transductionpathway.

Example 9: Cellular Interactions RGDKGE (SEQ ID NO: 1) ContainingPeptide Enhances Nuclear YAP Accumulation and Endothelial Cell Growth

The data indicate that while both RGD containing collagen peptides P-1and P-2 support an initial β3-integrin-mediated endothelial celladhesive interaction, collagen peptide P-2 selectively enhancedβ3-integrin phosphorylation leading to increased activation of P38MAPKand accelerated actin stress fiber formation. Actin stress fiberformation and enhanced mechanical tension contribute to re-localizationof the transcriptional co-activator Yes-associated protein (YAP) to thenucleus (46). Moreover, YAP is implicated in regulating angiogenesis andendothelial cell growth (47, 48). YAP localization following endothelialcell binding to the distinct collagen RGD containing peptides in theabsence of growth factor stimulation was examined. As shown in FIG. 8A,enhanced level of nuclear localized YAP was detected by 15 minutesfollowing endothelial cell binding to collagen peptide P-2 as comparedto P-1. The differential nuclear localization of YAP was notconsistently detected at later time points. To confirm this enhancednuclear accumulation of YAP, Western blot analysis was carried out. Asshown in FIG. 8B, the levels of nuclear YAP increased by approximately2-fold in endothelial cells attached to the peptide P-2 as compared topeptide P-1.

Effects of RGD Containing Collagen Peptides on HUVEC and HRMVEC Growth

Nuclear localization of YAP contributes to the regulation of endothelialcell growth. Therefore, the effects of RGD containing collagen peptideson HUVEC and HRMVEC growth were examined. Addition of soluble P-2significantly (P<0.05) enhanced endothelial cell growth whilestimulation with RGDAPG (SEQ ID NO: 11) peptide P-1 had a minimal effect(FIG. 8C and FIG. 8D). The selective ability of P-2 to enhanceSrc-dependent activation of P-38MAPK, the ability of the enhancednuclear accumulation of YAP following RGDKGE (SEQ ID NO: 1) peptide P-2stimulation was Src-dependent and/or P38MAPK dependent was assessed. Asshown in FIG. 8E and FIG. 8F, nuclear YAP accumulation following P-2stimulated endothelial cells was reduced by approximately 50% bytreatment with either Src or P38MAPK inhibitors as compared to control.These data are consistent with a role for Src and P38MAPK in mediatingthe RGDKGE (SEQ ID NO: 1) P-2 stimulated nuclear accumulation of YAP.

P-2 Stimulated Endothelial Cell Growth Depends on YAP

YAP plays roles in regulating the expression of multiple angiogenesisand inflammatory factors; therefore it was examined whether P-2stimulated endothelial cell growth depends on YAP. Endothelial cellswith YAP specific or non-specific shRNAs were transduced. Addition ofsoluble RGDKGE (SEQ ID NO: 1) containing collagen peptide P-2 toendothelial cells, but not the related RGDAPG (SEQ ID NO: 11) containingpeptide P1, (P<0.05) enhanced growth in control transduced cells (FIG.8G), while the RGDKGE (SEQ ID NO: 1) peptide P-2 failed to induce growthin endothelial cells in which YAP was knocked down (FIG. 8H). YAP knockdown cells were capable of proliferating as enhanced growth was observedfollowing VEGF or high serum stimulation (FIG. 8I). Together, thefindings are consistent with a mechanism by which RGDKGE (SEQ ID NO: 1)containing collagen peptide P-2 initiated a unique mechano-signalingcascade leading to the activation of P38MAPK in a Src-dependent pathwaythat ultimately led to nuclear YAP accumulation and enhanced endothelialcell growth.

Example 10: Enhanced Expression of PDL-1 in Human M21 Melanoma Tumors asCompared to M21L Melanoma Tumors Lacking αvβ3

Nude mice were injected with either M21 (αvβ3+) or ML21 (αvβ3−) melanomacells and were allowed to establish pre-existing tumors. Tumor sectionswere analyzed for expression of the immune checkpoint regulatory proteinPDL-1 by immunofluorescence staining (FIG. 10).

Example 11: Enhanced Expression of PDL-1 in Human M21 Melanoma Tumors asCompared to M21 Melanoma Tumors in which β3 Integrin was Knocked Down

M21 melanoma cells that express integrin αvβ3 were transfected withnon-specific control shRNA (M21 Cont) or β3 specific shRNA (M21β3 Kd)(FIG. 11). Nude mice were injected with either M21 Cont (αvβ3+) or M21β3Kd (αvβ3−) melanoma cells and were allowed to establish pre-existingtumors. Tumor sections were analyzed for expression of the immunecheckpoint regulatory protein PDL-1 by immunofluorescence staining.

Example 12: MabXL313 Enhanced the Anti-Tumor Activity of the ImmuneCheckpoint Inhibitor Anti-PDL-1 Antibody

Mice were injected with B16F10 melanoma cells and were allowed toestablish pre-existing tumors for 5 days prior to treatment (FIG. 9).Data in FIG. 9 represent mean tumor volume at day 14±SE from 8 mice percondition.

Example 13: Reduced Expression of PDL-1 in Melanoma Tumors in MiceTreated with an Antibody Targeting the αvβ3 Ligand (RGDKGE (SEQ IDNO: 1) Containing XL313 Collagen Epitope)

Mice were injected with melanoma cells and were allowed to establishpre-existing tumors for 5 days prior to treatment. Mice were treated 3times a week for 14 days with an antibody targeting the αvβ3 bindingRGDKGE (SEQ ID NO: 1) containing collagen epitope. Tumor sections wereanalyzed for expression of the immune checkpoint regulatory proteinPDL-1 by immunofluorescence staining (FIG. 12).

Example 14: Enhanced Detection of Lymphocytic Infiltrates in MelanomaTumors in Mice Treated with an Antibody Targeting the αvβ3 Ligand(RGDKGE (SEQ ID NO: 1) Containing XL313 Collagen Epitope)

Mice were injected with melanoma cells and were allowed to establishpre-existing tumors for 5 days prior to treatment. Mice were treated 3times a week for 14 days with an antibody targeting the αvβ3 bindingRGDKGE (SEQ ID NO: 1) containing collagen epitope (FIG. 13). Tumorsections were analyzed for expression of the immune checkpointregulatory protein PDL-1.

Example 15: Detection of Enhanced Levels of PDL-1 Protein in Melanomaand Endothelial Cells Following Binding to 3 ECM Ligand (DenaturedCollagen-IV)

Integrin αvβ3 expressing melanoma cells M21 (FIG. 14A) and CCL-49 (FIG.14B) and endothelial cells (HUVEC) (FIG. 14C) were attached to wellscoated with either a non-αvβ3 binding ligand (native collagen-IV) or anαvβ3 binding ligand (denatured collagen-IV). Whole cell lysates wereprepared and the relative levels of PDL-1 or β-actin loading controlwere assessed.

Example 16: Enhanced Levels of PDL-1 Protein in Melanoma Cells FollowingBinding to αvβ3 Ligand XL313 Epitope (RGDKGE (SEQ ID NO: 1))

Integrin αvβ3 expressing melanoma cells (M21) were seeded onto controluncoated wells or wells immobilized with the XL313 cryptic collagenepitope (RGDKGE (SEQ ID NO: 1)) (FIG. 15). Following an incubationperiod, cell lysates were prepared and relative levels of PDL-1 wereassessed.

Example 17: Reduction in the Levels of PDL-1 Protein in Melanoma CellsFollowing Blocking Binding to αvβ3 ECM Ligand (Denatured Collagen-IV)with a αvβ3 Specific Antibody LM609

Integrin αvβ3 expressing melanoma cells (M21) were mixed with a controlnon-specific (normal mouse Ig) or αvβ3 specific antibody (Mab LM609) andwas added to wells coated with αvβ3 binding ligand (denaturedcollagen-IV) (FIG. 16). Whole cell lysates were prepared following a 24hour incubation period and the relative levels of PDL-1 or loadingcontrol were assessed.

The following references were cited in Examples 1-17.

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Example 18: Inhibition of Ovarian Tumor Growth by Targeting the HU177Cryptic Collagen Epitope

Described herein are results demonstrating the anti-angiogenic andanti-tumor activity of antibody antagonists directed to the HU177cryptic collagen epitope. As described in detail below, antagonists ofthe HU177 epitope reduce stromal cell infiltration of tumors and inhibitangiogenesis and tumor growth.

As described in detail below, the HU177 epitope regulates ovarian tumorgrowth. Evidence suggests that stromal cells play critical roles intumor growth. As described herein, uncovering new mechanisms thatcontrol stromal cell behavior and their accumulation within tumors leadsto development of more effective treatments for malignant cancers.Described herein is evidence that the HU177 cryptic collagen epitope isselectively generated within human ovarian carcinomas and that thiscollagen epitope plays a functional role in SKOV-3 ovarian tumor growthin vivo. The ability of the HU177 epitope to regulate SKOV-3 tumorgrowth depends in part on its ability to modulate stromal cell behavioras targeting this epitope inhibited angiogenesis and surprisingly, theaccumulation of aSMA expressing stromal cells. As described in detailbelow, integrin α10β1 can serve as a receptor for the HU177 epitope inaSMA expressing stromal cells and subsequently regulates Erk-dependentmigration. The findings presented herein are consistent with a mechanismby which the generation of the HU177 collagen epitope provides apreviously unrecognized α10β1-ligand that selectively governsangiogenesis and the accumulation of stromal cells, which in turnsecrete pro-tumorigenic factors that contribute to ovarian tumor growth.Taken together, the findings presented herein provide new mechanisticunderstanding into the roles by which the HU177 epitope regulatesovarian tumor growth and provide new insight into the clinical resultsfrom a phase-I human clinical study of Mab D93/TRC093 in subjects withadvanced malignant tumors.

The importance of stromal cells such as endothelial cells, fibroblasts,pericytes and inflammatory infiltrates in tumor growth has beenappreciated for years (1-4). This insight has led investigators to begindeveloping novel approaches to regulate stromal cell behavior (5-8).However, given the functions of stromal cells in normal physiologicalprocesses, it is important to create strategies that might restrict theimpact on stromal cells to that within the tumor microenvironment. Inthis regard, the structures of extracellular matrix (ECM) proteins thatcompose the architectural framework of most normal tissues are largelyintact. In contrast, tumors often exhibit an altered ECM structure withproteolytically degraded matrix proteins (9, 10). As described herein,this differential ECM configuration provides a unique means forselectively regulating stromal cell behavior within tumors sincecellular interactions with remodeled or denatured matrix proteins suchas collagen alters adhesion, migration, proliferation and survival(11-14).

Previous studies uncovered functional cryptic sites within ECM molecules(14-16). As described herein, the process of generating cryptic elementsis likened to that of a biomechanical ECM switch, in which structuralalterations in these molecules initiated by either proteolytic cleavageor other physical mechanisms leads to the generation of crypticregulatory epitopes which contribute to the initiation of uniquesignaling cascades that facilitate angiogenesis, tumor growth andmetastasis (11-17). Recently, a new cryptic ECM epitope was identifiedwithin multiple forms of collagen (15). The HU177 epitope was generatedwithin the ECM of angiogenic vessels and regulates endothelial cellbehavior, as a monoclonal antibody directed to this epitope selectivelyinhibited endothelial cell adhesion and migration on denatured collagenand blocked angiogenesis in vivo (15). This antibody was humanized (MabD93/TRC093) and a phase-I human clinical trial was completed (18-20).Clinical findings suggested that the HU177 epitope plays a role in tumorgrowth as 26% of the treated subjects exhibited stable disease and areduction in liver lesions was observed in a subject with ovarian cancer(20).

Ovarian cancer is a heterogeneous disease classified by distincthistological subtypes (21-25). The molecular complexity of these tumorsis demonstrated by the fact that low-grade type-I tumors often exhibitalterations in KRAS, BRAF and PTEN, while high-grade type-II tumorsoften have alterations in TP53 and BRCA1/2 (21-25). Importantly, stromalcells such as endothelial cells and activated fibroblasts may contributeto the development of ovarian carcinoma (26-28). Prior to the inventiondescribed herein, while collagen remodeling occurs during ovarian tumorgrowth, it was not known whether these changes are sufficient togenerate the HU177 epitope or what role it plays in ovarian tumorgrowth.

Described herein is evidence that the HU177 cryptic collagen epitope isabundantly generated within human ovarian tumors, while little isexpressed in benign granulomas. As described in detail below, antibodiesdirected to this epitope inhibited SKOV-3 tumor growth in vivo, whichwas accompanied by reductions in proliferation, angiogenesis and theaccumulation of aSMA expressing stromal cells. While these studiesindicate that α2β1 integrin can bind the HU177 site, the littleunderstood integrin α10β1 plays an important role as a functionalreceptor in aSMA-expressing stromal cells. Blocking interactions of theHU177 collagen epitope with α10β1 integrin expressing fibroblastsreduced FGF2-stimulated Erk phosphorylation and migration on denaturedcollagen. Given the emerging roles of fibroblast-like cells in promotingtumor growth, these findings are consistent with a mechanism by whichblocking the HU177 epitope reduces α10β1-dependent accumulation of aSMAexpressing stromal cells in ovarian tumors, leading to the reduction ofan important source of pro-tumorigenic cytokines that contribute totumor progression.

The following Materials and Methods were utilized for Example 18.

Reagents, Chemicals and Antibodies.

Collagen type-I was from Sigma (St Louis, Mo.). Denatured collagen wasprepared by boiling the solution of commercially obtained collagen for15 minutes. The denatured collagen was allowed to cool for 5 minutesprior to use. Fibroblast growth factor-2 (FGF-2), integrins α1ß1, α2ß1,α3ß1, α10ß1, αvß3 and antibodies directed to α1, α2, αv integrins andIL-6 were from R&D Systems (Minneapolis, Minn.). Anti-CD-31 antibody wasfrom BD Pharmingen (San Diego, Calif.). Anti-αSMA and anti-Ki67antibodies were from Abcam (Cambridge Mass.). Anti-Erk antibodies werefrom Cell Signaling Technology (Danvers, Mass.). Anti-collagen-Iantibody was from Rockland (Gilbertsville, Pa.). Antibody to α10ß1 wasfrom Novus Biologicals (Littleton Colo.). BrdU kit was from Millipore(Bedford, Mass.).

Secondary antibodies were from Promega (Madison, Wis.). Mab HU177 wasdeveloped in a laboratory and shown to bind a PG×PG containing epitopesexposed within denatured, but not intact collagen from multiple species(11-16). Mab D93/TRC093 is a humanized version of Mab HU177 that alsobinds the PG×PG containing epitopes within denatured collagen frommultiple species and was obtained from TRACON (San Diego, Calif.). Thecontrol antibody (Mab XL166) was generated in a laboratory and isdirected to an RGD collagen epitope. Collagen PGF-peptide (CPGFPGFC; SEQID NO: 16) and Cont-peptides (CQGPSGAPGEC; SEQ ID NO: 10),(CTWPRHHTTDALL; SEQ ID NO: 17) and (CNSYSYPSLRSP; SEQ ID NO: 18) werefrom QED Biosciences (San Diego Calif.). MEK inhibitor (PD98059) wasfrom CalBoichem (San Diego, Calif.).

Analysis of Tissue Antigens.

Human ovarian tissues were from MMC under IRB exempt protocol (No.3175x). For quantification of the HU177 epitope, biopsies (N=9) fromhigh-grade ovarian tumors (serous and endometrial) or benign ovariangranulomas (N=9) were stained with Mab HU177. OCT compound embeddedfrozen sections (4.0 um) of biopsies of human ovarian tumor tissues wereeither stained by routine hematoxylin and eosin (H&E) procedure or byimmunofluorescence. Immunofluoresence staining was performed on frozensections by first blocking the tissue sections with 1.0% BSA in PBS for1 hr followed by washing 3× with PBS. Tissue sections were nextincubated with anti-HU177 antibody (100 ug/ml) for 1 hour at roomtemperature. Tissues sections were next washed 3× with PBS followed byincubation with FITC-labeled secondary antibody for 1 hr. Forquantification, stained tissue sections were scanned using Kodak IDsystem and pixel density quantified from five 200× fields from each of 5specimens from each condition using Molecular Analyst Software ver 2.1(29). Tumors were analyzed for apoptosis using TUNEL staining, forproliferation using anti-Ki67 antibody staining (1:1000), forangiogenesis using anti-CD31 antibody staining (1:300) and for CAF-likestromal cells using a combination of anti-aSMA (1:1000), anti-PDGF-Ra(1:500) and anti-FAP (1:300) antibody staining. Quantification wasperformed within 5, 200× fields from each of 3 to 5 tumors.

Cells and Cell Culture.

SKOV-3 cells, were from ATCC (Manassas, Va.) and cultured in RPMI in thepresence of 5% FBS. Human umbilical vein endothelial cells (HUVECs) wereobtained from Lonza (Walkersville, Md.) and cultured in EBM-2 medium in2% FBS and supplements (Lonza). Human dermal fibroblasts were obtainedfrom Science Cell (Carlsbad, Calif.) and cultured in medium with 2.0%FBS.

Solid-Phase Binding Assays.

Plates were coated with 25 μg/ml of native or denatured (boiled 15minutes) collagen.

Collagen epitope peptide PGF and Cont-peptides were immobilized at 100ug/ml. Integrins (0-2.0 μg/ml) were diluted in binding buffer containing20 mM Tris, 15 mM NaCl, 1 mM MgCl2, 0.2 mM MnCl2, 0.5% BSA pH 7.4 asdescribed (13). Integrins were allowed to bind for 1 hr and plateswashed and incubated with anti-integrin antibodies (1:100 dilution) for1 hr. Plates were washed and incubated with HRP-labeled secondaryantibodies (1:5000 dilution). In a second assay, integrins (0.5 μg/ml)were coated in binding buffer and plates were washed, blocked as beforeand denatured (boiled 15 minutes) collagen was added (0-10.0 μg/ml).Denatured collagen was detected with anti-collagen antibody (1:1000).For integrin blocking ELISAs, wells were coated with denatured collagenand pre-treated (0.1p g/ml) with anti-HU177 or control antibody.Following washing, wells were incubated with integrins (2.0 μg/ml) andbinding was detected as described. Assays were carried out at least 3times.

Cell Adhesion Assays.

The wells of non-tissue culture forty-eight well cluster plates werecoated for 12 hrs at 4° C. with 5 μg/ml of native or denatured (boiled15 minutes) collagen prepared as described above. SKOV-3 and fibroblastswere suspended in adhesion buffer containing RPMI 1640, 1 mM MgCl2, 0.2mM MnCl2 and 0.5% BSA (13) and 1×105 cells were added in the presence orabsence of anti-HU177 or control antibodies (100 μg/ml). Cells wereallowed to attach for 25 minutes. Non-attached cells were removed bywashing and attached cells stained with crystal violet as described(13). Wells were washed and the cell-associated crystal violet waseluted with 100 ul per well of 10% acetic acid. Cell adhesion wasquantified by measuring the optical density of the eluted crystal violetas described (15). Assays were completed at least 3×.

Cell Migration Assays.

Transwell membranes (8.0 um pore size) were coated with 5 μg/ml ofnative or denatured collagen for 12 hrs at 40 C as described previously(13). Migration buffer containing RPMI 1640, 1 mM MgCl2, 0.2 mM MnCl2and 0.5% BSA in the presence or absence of 5× concentrated serum freeSKOV-3 CM or 20 ng/ml FGF-2 was placed in the lower chambers. SKOV-3 orfibroblasts (1×105) were resuspended in migration buffer containing RPMI1640, 1 mM MgCl2, 0.2 mM MnCl2 and 0.5% BSA in the presence or absenceof antibodies directed to the HU177 epitope, or α10β1, (10 μg/ml) or 100uM of the MEK inhibitor (PD98054). Cells were added to the top chamberand allowed to migrate from 2 to 4 hrs. Cells remaining on the top ofthe membranes were removed and cells that had migrated to the undersideof the membrane were quantified by direct cell counts of crystal violetstained cells or by measuring the optical density of elutedcell-associated crystal violet dye as previously described (15). Assayswere completed at least 3×.

Cell Proliferation Assays.

To prepare serum free fibroblast CM, 2.0×10⁶ cells were seeded in thepresence of serum for 2 hrs. Serum containing medium was removed andcells were washed and serum free RPMI was added. Serum free medium wascollected at 24 hrs and concentrated 10-fold. SKOV-3 cells wereresuspended in the presence (150 μl) of control (10×) RPMI or (10×)fibroblast CM. Cells were added to wells and allowed to growth for 24 or72 hrs. Cell proliferation was quantified at 24 or 72 hrs using BrdU kitaccording to manufactures instructions. Assays were completed 3 times.

Tumor Growth Assays.

Briefly, CAMs of 10-day chicks (N=6-8) were prepared as described (13)by separating the CAM from the shell membrane. Single cell suspensionsof SKOV-3 cells (3×10⁶) were applied topically to the CAMs in a totalvolume of 40 uls (30). Twenty-four hours later embryos were eitheruntreated or treated with a single topical application of 100 ugs ofanti-HU177 antibody or control non-specific antibody (100 ug/embryo) ina total volume of 40 ul. Tumors were allowed to grow for a total of 7days. At the end of the 7-day growth period the embryos were sacrificedand the tumors dissected and wet weights determined as describedpreviously (13). For murine experiments, nude (NCRNU-F) mice (N=6-8)were injected subcutaneously into the flanks with SKOV-3 cells(3.0×10⁶/mouse). Mice were allowed to form tumors for 5 days and thenuntreated or injected (i.p) with Mab D93 or control antibody (0-100μg/ml) 3× per week for 28 days. Tumors were measured with calipers andvolumes calculated using the formula V=L2×W/2 were V=volume, L=length,W=width. Experiments were completed 3×.

Western Blot Analysis.

Cells from culture or cells seeded on native or denatured (boiled for 15minutes) collagen type-I coated plates were allowed to attach for 15minutes then were lysed in RIPA buffer (Santa Cruz, Calif.) with 1×protease inhibitor cocktail. Lysates were separated by SDS PAGE andprobed with antibodies directed to α10, or α1 integrins, total andphosphorylated Erk, Ki67 and β-tubulin or actin. Assays were performedat least 3 times.

Cytokine Analysis of Fibroblast CM.

Serum free CM (50 μl) was screened for a panel of 12 cytokines (IL-1α,IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17A, TNF-α, INF-γ,GM-CSF) using the Multi-analyte ELISArray™ kit (Qiagen) according tomanufactures instructions. Analysis of conditioned medium was completedtwice.

Statistical Analysis.

Statistical analysis was performed using the InStat statistical program.Data analyzed for significance using Student T test. P values <0.05 wereconsidered significant.

Results Differential Generation of the HU177 Epitope in Ovarian Tumors.

Studies have correlated enhanced collagen synthesis and ECM degradationwith tumor progression (31, 32). In this regard, biopsies of humanovarian tumors and benign granulomas were examined for expression of theHU177 cryptic collagen epitope. Serial sections from frozen tissues werestained by H&E or with anti-HU177 antibody. As shown in FIG. 17A, theHU177 cryptic collagen epitope (green) could be detected in theextracellular matrix (ECM) of malignant ovarian tumors while minimaldetection was observed in the ECM of the benign ovarian lesions (FIG.17B). Quantification of the relative levels of the HU177 crypticcollagen epitope (FIG. 17C) indicated a significant (P<0.05) increase inHU177 collagen epitope in tumors as compared to the benign lesions. Toconfirm the generation of the HU177 epitope in an experimental mousemodel, its expression of the HU177 collagen epitope was examined inSKOV-3 tumors growing in mice. As shown in FIG. 17D, while HU177 epitopewas detected within the SKOV-3 tumors, little was observed in normaltissues including ovaries, liver and lungs. These findings areconsistent with the restricted generation of the collagen epitope.

The HU177 Epitope Regulates Ovarian Tumor Growth.

Given the differential generation of the HU177 epitope observed in vivo,it was determined whether the HU177 epitope plays a functional role inovarian tumor growth. To examine this possibility, SKOV-3 ovarian tumorcells were seeded topically on the chorioallantoic membrane (CAM) ofchick embryos. Twenty-four hours later, the embryos were either nottreated or treated with a single topical application with anti-HU177antibody or non-specific control. Following 7 days of incubation, theresulting tumors were dissected from the CAMs for analysis. As shown inFIG. 182A, SKOV-3 tumors dissected from chick embryos treated withanti-HU177 antibody were smaller than controls. Quantification indicatedthat targeting the HU177 epitope significantly (P<0.05) inhibited SKOV-3tumor growth by approximately 50% as compared to controls (FIG. 18B). Toconfirm these findings in a murine model, SKOV-3 cells were injectedsubcutaneously into the flanks of nude mice. Five days later followingestablishment of small tumor lesions, mice were either untreated ortreated (100 ug/mouse 3× per week) intraperitoneally (i.p) withanti-HU177 Mab or control non-specific antibody. As shown in FIG. 18C,treatment with anti-HU177 antibody significantly (P<0.05) reduced tumorsize by approximately 70% by day 28. Dose dependent studies indicatedinhibition of tumor growth at doses of anti-HU177 antibody as low as 10μg/mouse 3 times per week (data not shown).

The HU177 Epitope Regulates Angiogenesis and Stromal Cell Accumulationin Ovarian Tumors.

The behavior of multiple cell types within the tumor microenvironmentincluding stromal cells can be controlled by interactions with collagen(2,6,33-37). Therefore, apoptosis was examined within control andanti-HU177 treated SKOV-3 tumors using TUNEL staining. An approximately1.8 fold increase in apoptosis was detected within anti-HU177 treatedtumors as compared to control, however, due to variation in staining,this increase did not meet statistical significance. Next, cellularproliferation was examined by quantifying Ki67 antigen expression withinthese tumors growing in vivo. As shown in FIG. 19A, SKOV-3 tumors fromanti-HU177 Mab treated mice exhibited significantly (P<0.05) reduced(25%-40%) Ki67 expression as compared to no treatment or controlantibody, respectively. Given previous studies indicating that blockingthe HU177 epitope selectively inhibited endothelial cell adhesion todenatured collagen type-IV but not native intact collagen type-IV (15)and inhibited angiogenesis in vivo (15, 18), the reduction in cellularproliferation within these tumors could be due to multiple mechanismsincluding inhibition of angiogenesis given the known role ofangiogenesis in regulating tumor growth.

To examine the distribution of the HU177 epitope in ovarian tumors,SKOV-3 tumors were co-stained with antibodies directed to the HUI77epitope and CD-31 a marker of blood vessels. While the HU177 epitope wasdetected in association with some CD-31 positive vessels (FIG. 19B, top)it was more extensively associated with αSMA expressing fibroblast-likestromal cells (FIG. 19B bottom). Given the distribution of the HU177epitope in the vicinity of blood vessels and aSMA expressing stromalcells, the effects of anti-HU177 Mab treatment on SKOV-3 tumorangiogenesis and the accumulation of tumor-associated αSMA expressingcells was further analyzed. As shown in FIG. 19C, a significant (P<0.05)35% inhibition of SKOV-3 ovarian tumor angiogenesis was observed andremarkably, a 70% reduction in αSMA expressing stromal cells was alsodetected following treatment with anti-HU177 antibody (FIG. 19D).

These findings are consistent with the notion that reduced angiogenesisand αSMA expressing stromal cell accumulation may contribute to theanti-tumor activity observed following selective blockade of the HU177collagen epitope.

SKOV-3 Tumor Cells and Fibroblast Exhibit Enhanced Adhesion andMigration on Denatured Collagen.

Previous studies have documented that changes in the native structure ofECM proteins can alter the ability of cells to bind to these denaturedproteins in part by disrupting conformational dependent epitopesrequired for cell binding in the context of its native configurationand/or exposing previously hidden cryptic binding epitopes includingcryptic RGD containing sequences (12-16). However, the ability ofdistinct cell types to interact with denatured collagen may vary widelyand likely depends on multiple factors including the expression andactivation of cell surface receptors with the capacity to recognize andbind to the multiple cryptic epitopes exposed following denaturation. Toexamine whether cellular interactions with denatured collagen alterscellular behavior, SKOV-3 and fibroblast adhesion and migration onnative and denatured collagen was compared. As shown in FIGS. 20A and20B, a small but significant (P<0.05) enhancement of SKOV-3 (38%) andfibroblast (29%) adhesion was detected on denatured collagen as comparedto native collagen. In similar studies, the basal migratory capacity ofSKOV-3 and fibroblasts on native and denatured collagen was compared. Asshown in FIGS. 20C and 20D, again a small but significant (P<0.05)enhancement of SKOV-3 (34%) and fibroblast (19%) migration was detectedon denatured collagen as compared to native collagen. While thesestudies do not specifically indicate that the enhanced adhesion andmigration of the cells on denatured collagen depends specifically on theHU177 epitope, given that multiple cryptic epitopes are exposedfollowing denaturation, these observations, however, do indicate thatinteractions with denatured collagen can alter the behavior of SKOV-3cells and fibroblasts in vitro.

The HU177 Epitope Regulates SKOV-3 and Fibroblast Adhesion and Migrationon Denatured Collagen.

Tumor and fibroblast adhesive interactions with denatured collagen,which has been shown to be present within the microenvironment ofmalignant cancers, may regulate tumor progression. Given studiesindicating that denaturation of collagen can alter the adhesive andmigratory behavior of SKOV-3 cells and fibroblasts, the role of theHU177 collagen epitope on regulating adhesion and migration of thesecells was examined. As shown in FIG. 21A, selectively blocking the HU177epitope exposed within denatured collagen, significantly (P<0.05)inhibited adhesion of SKOV-3 cells to denatured collagen byapproximately 40%. In contrast, the anti-HU177 antibody failed toinhibit adhesion to intact collagen (FIG. 21B). These data areconsistent with previous studies indicating a selective inhibition ofcellular interactions with denatured collagen given that the anti-HU177antibody does not bind native intact collagen (15-18). In similarstudies, the effects of targeting the HU177 epitope on fibroblastadhesion to denatured collagen was examined. Similar to what wasobserved with SKOV-3 tumor cells, blocking the HU177 epitopesignificantly (P<0.05) inhibited fibroblast adhesion to denaturedcollagen by approximately 50% as compared to controls while thisantibody had no effect on fibroblast adhesion on native intact collagen(FIGS. 21C and 21D). These data indicate the ability of the anti-HU177antibody to inhibit adhesion is restricted to those circumstances inwhich the HU177 epitope is generated and thus the anti-HU177 antibodydoes not alter adhesion to intact collagen thereby allowing a highlyselective strategy to inhibit adhesive cellular interactions withdenatured collagen.

Next, the migratory capacity of these cells on native and denaturedcollagen was examined. As shown in FIG. 21E through 21F, blocking theHU177 epitope exposed within denatured collagen also significantly(P<0.05) inhibited basal migration of SKOV-3 and fibroblasts by 35% to38%, while migration on intact collagen was not effected (FIG. 21E-21H).Interestingly, while blocking the HU177 epitope inhibited adhesion andmigration, it exhibited little effects on the growth of SKOV-3 cells orfibroblasts on denatured collagen when examined in vitro. These studiesare consistent with the anti-HU177 antibody having minimal if any directanti-proliferative activity on fibroblasts or SKOV-3 cells on denaturedcollagen in vitro.

Inhibition of Growth Factor-Induced Fibroblast Migration by Targetingthe HU177 Epitope.

Accumulating evidence indicates that recruitment of activated and/orcancer-associated fibroblasts may facilitate tumor growth and survival,in part by providing an important source of pro-tumorigenic factorsincluding IL-6 (2, 5, 6, 38). While it is clear that most dermalfibroblast do not represent a fully accurate model of cancer associatedstromal cells found in vivo, following in vitro culture, the fibroblastsexhibited phosphorylated Erk and expressed αSMA and PDGF-Rα, importantmarkers of CAF-like stromal cells. Therefore, these fibroblasts wereused as a model of αSMA expressing stromal cells and it was examinedwhether blocking the HU177 epitope could alter growth factor-inducedmigration. As shown in FIGS. 22A and 22B, concentrated SKOV-3 CM as wellas recombinant FGF-2 stimulation enhanced migration on denaturedcollagen. Importantly, addition of the anti-HU177 antibody to themigration wells significantly (P<0.05) inhibited this growth factorstimulated response suggesting that not only can the HU177 epitope playa role in the basal cell migration, but also help regulate growth factormediated motility.

Previous studies have indicated that FGF-2 stimulation can initiate acomplex signaling cascade leading to the phosphorylation of multipleeffector molecules and transcription factors such as Erk1/2, which helpcoordinate the complex events required for cellular migration (39-44).Given the known role of FGF-2 in regulating Erk activation, the effectsof blocking fibroblast interactions with the HU177 epitope on FGF-2stimulated Erk phosphorylation were examined. As shown in FIG. 22C,FGF-2 stimulated a small increase in Erk phosphorylation in cellsattached to denatured collagen, while blocking the HU177 epitope reducedthis phosphorylation. A mean reduction in Erk phosphorylation from 4independent experiments of approximately 40% was observed (FIG. 22D). Toconfirm a role for MAP/Erk signaling in FGF-2 stimulated fibroblastmotility on denatured collagen, migration assays were carried out in thepresence of a MEK inhibitor. As shown in FIG. 22E, addition of the MEKinhibitor (PD98059) significantly (P<0.05) blocked FGF-2 stimulatedmigration on denatured collagen suggesting a role for MAP/Erk signalingin regulating this migratory response.

Identification of Receptors for the HU177 Epitope.

The repetitive PG×PG containing sequence as a critical component of theHU177 epitope was previously identified (15). In addition, other studieshave indicated that antibodies directed to the HU177 epitope can bind toa variety of distinct PG×PG containing sequences with differentN-terminal and C-terminal flanking sequences found in collagen (19).Given that a number of variations of this motif are found throughoutcollagen, it is possible that multiple receptors may have the ability tobind to these cryptic sites. To this end, integrin receptors are animportant class of molecules known to mediate cellular interactions withECM components and coordinate signaling cascades that govern cellmotility. To define potential cell surface receptors for HU177 collagenepitope, the ability of various recombinant integrins to bind denaturedcollagen was examined. Integrin α2β1 and αvβ3 dose dependently bounddenatured collagen, while α1β1 showed minimal interactions (FIG. 23A).Interestingly, integrin α10β1, a collagen binding receptor thought tohave a limited tissue distribution outside of cartilage (45-48) alsodose dependently bound denatured collagen. To confirm α10β1 binding todenatured collagen we carried out a second binding assay by immobilizingα10β1 on microliter wells and examining soluble denatured collagenbinding. Denatured collagen dose dependently bound α10β1, but not 301(FIG. 23B). To identify which integrins capable of binding denaturedcollagen have the ability to bind the HU177 epitope, the effects ofblocking the HU177 epitope on integrin binding were examined. As shownin FIG. 23C, blocking the HU177 epitope inhibited α10β1 binding bynearly 60%, while α2β1 binding was reduced by approximately 30% (FIG.23D). Binding of αvβ3 to denatured collagen was unaffected (FIG. 23E).To confirm the integrin binding specificity, direct binding to thesynthetic PG×PG containing HU177 epitope (PGF peptide) was performed. Asshown in FIG. 23F, while α2β1 bound the HU177 epitope, α10β1 exhibited4-fold greater binding, while αvβ3 failed to bind. Moreover, thePGF-peptide also partially competed binding of α10β1 to denaturedcollagen by approximately 60% (FIG. 23G). Taken together, these findingssuggest that the little understood integrin α10β1 can bind the HU177collagen epitope.

Integrin α10β1 Co-Localizes with αSMA Expressing Cells in OvarianTumors.

Given the studies indicating the ability of α10β1 to bind the HU177epitope, the expression of α10β1 within ovarian tumors was examined. Asshown in FIG. 24A, α10β1 was detected within human ovarian tumors (left)and SKOV-3 tumors (right). Expression of α10β1 was associatedpredominately with αSMA positive cells as suggested by extensiveco-localization (FIG. 24B).

While minimal levels of α10 integrin protein (FIG. 24C) and mRNA weredetected in SKOV-3 cells, greater than 5 fold higher levels weredetected in αSMA expressing fibroblasts. Next, the effects of blockingα10β1 on fibroblast migration were examined. As shown in FIG. 24D,anti-α10 antibody inhibited FGF-2 stimulated migration on denaturedcollagen. To confirm a role for α10β1 integrin in fibroblast migrationon denatured collagen, expression α10β1 was integrin knocked down inthese fibroblasts using α10 specific shRNA. As shown in FIG. 24E, therelative levels of α10 protein were reduced by approximately 70% whilelittle change in α1 integrin chain or actin was observed. Next, theability of fibroblasts expressing altered levels of α10 integrin tomigrate on denatured collagen was examined. As shown in FIG. 24F, whilelittle if any change was observed in control transfected (Con-Kd-HF)fibroblasts as compared wild type parental cells (WT-HF), knocking downα10 integrin significantly (P<0.05) inhibited fibroblast migration ondenatured collagen by approximately 60% as compared to controls.

Given these results, it's possible that the reduction of αSMA positivestromal cells observed following anti-HU177 treatment leads to areduction in an important source of pro-tumorigenic cytokines thatfacilitate tumor growth and angiogenesis. To this end, it was examinedwhether CM from αSMA expressing fibroblasts might contain solublefactors that enhance SKOV-3 cell growth. As shown in FIGS. 24G and 24H,fibroblast CM enhanced proliferation and Ki67 antigen expression inSKOV-3 cells. These results are consistent with the ability of solublefactors released into the conditioned medium of these α10β1-expressingfibroblasts to enhance SKOV-3 tumor cell proliferation in vitro. Tocharacterize potential pro-tumorigenic factors expressed by theseαSMA-positive fibroblasts, concentrated serum free CM was screened usinga multianalyte cytokine array for expression of 12 different cytokines.Among the cytokines that were expressed at the highest levels was IL-6(Table 2).

Table 2 shows the analysis of cytokine expression in fibroblastsconditioned medium. Serum free conditioned medium (CM) was collectedfollowing a 24-hour incubation of α10β1 expressing human fibroblasts.Conditioned medium was concentrated 10 fold. Serum free 10× concentratedconditioned medium was examined for the presence of 12 differentcytokines using the Multi-analyte ELISArray™ detection kit. Data valuesrepresents optical density (O.D) readings at a wavelength of 450 nm fromthe human fibroblast conditioned medium sample (HFB), negative controlbuffer (Neg Cont) or Positive control samples provided in the assay kit(Pos Cont) for the expression of each of the 12 cytokines.

TABLE 2 HFB Neg Pos Cytokine (CM) Cont Cont IL-1α 0.471 0.058 1.857IL-1β 0.491 0.060 2.607 IL-2 0.759 0.066 0.707 IL-4 0.692 0.026 1.448IL-6 1.554 0.052 0.606 IL-8 3.785 0.043 3.292 IL-10 0.678 0.043 1.440IL-12 0.877 0.053 1.708 IL-17A 2.747 0.054 1.670 IFNγ 1.409 0.045 1.089TNF-α 0.435 0.040 0.564 GMCSF 2.194 0.043 2.966

Interestingly, IL-6 has been shown to enhance growth of ovariancarcinoma cells (49, 50) and thus, it was examined whether thispro-tumorigenic cytokine played afunctional role in mediating theability of αSMA expressing fibroblast CM to stimulate proliferation ofSKOV-3 tumor cells. As shown in FIG. 24I, fibroblast CM significantlyenhanced SKOV-3 tumor cell proliferation and a function-blockingantibody directed to IL-6 inhibited this response. These data areconsistent with a role for IL-6 expression from fibroblasts in promotingSKOV-3 tumor cell growth. Taken together, these findings suggest thepossibility that αSMA-expressing fibroblasts may serve as an importantsource of protumorigenic factors that facilitate SKOV-3 tumor growth andthat selectively reducing accumulation of αSMA expressing stromal cellsby inhibiting the HU177 collagen epitope contributes to the potentanti-tumor activity observed in vivo.

DISCUSSION

The array of mechanisms by which stromal cells such as endothelial cellsand activated αSMA expressing fibroblasts govern the malignant phenotypeare diverse and include providing proteolytic enzymes that alter thebiomechanical properties of ECM, the release of protumorigenic factorsthat act on both tumor and other cell types to alter their growth,survival and migratory behavior, and the release of molecules thatinfluence the host immune response (1-6, 51, 52). Thus, selectivelyblocking the accumulation of pro-tumorigenic stromal cells in malignantlesions likely represents an important therapeutic strategy. Whiletargeting stromal cells may provide a complementary strategy for tumortherapy, prior to the invention described herein, it was challenging toselectively target them without disrupting their activity in normaltissues. The ECM represents an active control point for multiplemechanisms critical for regulating stromal cell behaviors ranging frommigration and proliferation to gene expression (14). Therefore,inhibiting stromal cell-ECM interaction that selectively limit theaccumulation of pro-tumorigenic stromal cells in malignant lesions mightrepresent a useful strategy to control tumor progression.

Here we show that the HU177 collagen epitope is abundantly generatedwithin ovarian carcinomas as compared to benign ovarian lesions.Importantly, previous studies have indicated that that proteolyticenzymes such as MMPs can contribute to generation of the HU177 epitopein vivo (16). As described herein, this cryptic collagen epitope playeda role in SKOV-3 tumor growth and these findings are consistent with aclinical trial assessing tolerability and toxicity of Mab D93/TRC093(20). Results from this study suggested no dose limiting toxicities andevidence of anti-tumor activity as a subject with an ovarian cancershowed a reduction in metastatic liver lesions (20). While all subjectseventually progressed, 26% exhibited disease stabilization and a subjectwith hemagiopericytoma, a tumor known to be highly infiltrated withstromal cells exhibited stable disease for nearly a year (20).

Consistent with previous findings (15, 18), the anti-HU177 antibodyinhibited tumor associated angiogenesis. These data are in agreementwith previous studies indicating that targeting the HU177 epitope couldselectively inhibit endothelial cell adhesion and migration on denaturedcollagen, inhibit proliferation, upregulate expression of P27KIP1 andinhibit angiogenesis in vivo (15). Surprisingly, the results presentedherein indicate that targeting the HU177 epitope can also reduce theaccumulation of αSMA expressing stromal cells within SKOV-3 tumors. Asdescribed herein, targeting the HU177 epitope inhibited SKOV-3 andαSMA-expressing fibroblast adhesion and migration on denatured but notintact collagen, thereby specifically limiting the impact of thistherapeutic agent to those tissues expressing the HU177 epitope. FGF-2induced fibroblast migration on denatured collagen was shown to bedependent in part on MAP/Erk signaling as inhibition of MAP/Erksignaling blocked this migratory response.

Cell migration is governed by a complex and integrated set of signalingevents that are coordinated in part by the unique composition andintegrity of the local extracellular matrix. However, prior to theinvention described herein, the mechanisms by which cells sense thestructural changes in the integrity of a given ECM environment duringtumor growth to facilitate cellular motility induced by growth factorswere not completely understood. In fact, inappropriate and/or enhancedmigration may contribute to tumor cell invasion and metastasis as wellas the accumulation of CAF-like stromal cells that contribute to tumorprogression. Integrin mediated interactions with ECM proteins are knownto initiate assembly of a multiple-protein signaling complexes thatcoordinately regulate multiple downstream signaling cascades includingShc/Grab2/Ras and Fak/Src/Rap1 pathways that can activate MAPK/Erksignaling (41). The results presented herein suggest FGF-2 can enhancethe phosphorylation of Erk in α10β1-expressing fibroblasts and afunction blocking antibody directed to the HU177 cryptic collagenepitope inhibited Erk phosphorylation. Given that MAPK/Erk signalingplays a role in the ability of α10β1-expressing fibroblasts to migrateon denatured collagen and the fact that denatured collagen has beenshown to be selectively generated within the tumor microenvironment (12,13, 15, 18), it would be interesting to speculate that the selectivegeneration of the HU177 epitope within the ovarian tumormicroenvironment may help facilitate enhanced activation of Erk whichmay promote stromal cell accumulation. While multiple protein kinasesare thought to contribute to the phosphorylation of Erk, the precisemolecular mechanism by which cellular interactions with the HU177epitope regulates FGF-2 induced activation of Erk were not known priorto the invention described herein. Studies are currently underway toconfirm which kinases and/or phosphatases might contribute to the FGF-2mediated regulation of Erk following interactions with the HU177collagen epitope.

Given the reduction in αSMA expressing cells in tumors treated withanti-HU177 antagonists, selectively inhibiting migration of thispopulation of stromal cells may limit a major cellular source ofpro-tumorigenic factors that play multiple roles in facilitating tumorcell survival and proliferation in vivo. In this regard, fibroblasts areknown to express many pro-tumorigenic factors including IL-6, which hasbeen previously shown to enhance proliferation of ovarian carcinomacells (38, 49, 50). Consistent with these findings, the resultspresented herein suggest that blocking IL-6 inhibited fibroblastCM-induced SKOV-3 cell growth. Given these findings and the high levelsof IL-6 expressed by fibroblasts, this data is consistent with amechanism by which targeting the HU177 epitope selectively limitsaccumulation of αSMA fibroblasts thereby reducing an important source ofpro-tumorigenic factors that enhance angiogenesis and tumor growth.

Described herein is new evidence that the little understood integrinα10β1 functions as a receptor for the HU177 collagen epitope infibroblasts. Given the variations within the HU177 PG×PG consensus siteand the possibility of unique geometrical configurations and distinctflanking amino acid sequences displayed in vivo, it is possible thatadditional receptors may also recognize the HU177 epitope.Interestingly, little is known concerning the functions of α10β1 outsideof chondrocyte and growth plate development (45-48). However, α10 mRNAhas been detected in murine heart, muscle tissues and endothelial cells(45-48). Studies suggest that FGF-2 stimulation of mesenchymal stemcells (MSC) leads to upregulation of α10 (53). Moreover, MSC have alsobeen implicated as potential sources of αSMA expressing CAF-like cellsin tumors (54). In addition, studies now suggest enhanced expression ofα10β1 in melanoma cell lines as compared to primary melanocytes andinhibiting α10β1 in these cells resulted in a reduced migration (55).

Given the ability of α10β1 to bind the HU177 epitope, the data presentedherein are in agreement with a mechanism by which generation of theHU177 epitope provides a previously unrecognized ligand forα10β1-expressing stromal cells that facilitates FGF-2 induced activationof Erk and subsequently the accumulation of αSMA positive stromal cellsin ovarian tumors. In turn, the selective reduction in accumulation ofthis cell population by anti-HU177 antibody, in conjunction with itspreviously described anti-angiogenic effects, likely contribute to itspotent anti-tumor activity. Taken together these findings provide newcellular and molecular insight into the roles of the HU177 crypticepitope in ovarian tumor growth and provide new mechanisticunderstanding of the therapeutic impact observed in human subjectstreated with Mab D93 (20).

The following references were cited in Example 18.

REFERENCES

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Example 19: The HU177 Collagen Epitope is Recognized by Integrin α10β1which Regulates Expression of the Immune Suppressive Cytokine IL-10

FIG. 25A is a bar chart wherein a peptide (cPG) containing the HU177collagen epitope CPGFPGFC (SEQ ID NO: 16) was immobilized on microtiterwells and the ability of recombinant integrin receptors to bind wasanalyzed by ELISA. Data bars represent mean integrin binding to theHU177 collagen epitope. FIG. 25B-FIG. 25D is a series of bar chartsshowing that the expression of α10 integrin chain in human C8161melanoma cells was reduced by transfection of shRNA directed to the α10integrin chain. Twenty-four hour serum free conditioned medium from eachmelanoma variant was concentrated 10× and analyzed for the relativelevels of cytokines. FIG. 25B is a bar graph wherein data bars representrelative levels of IL-10 as determined by ELISA. FIG. 25C is a bar graphwherein data bars represent relative levels of IL-2 as determined byELISA. FIG. 25D is a bar graph wherein data bars represent relativelevels of IL-4 as determined by ELISA. Thus, these results demonstratethat knocking down expression of an integrin receptor (Integrin α10β1),which can function as a receptor for the HU177 epitope in melanoma cellsreduced expression of a potent immune suppressive molecule IL-10.

Example 20: The Injection of a Peptide (cPG) Containing the HU177Collagen Epitope Induces Expression of the Immune Suppressive GrowthFactor TGF-β in Mouse Circulation

C57BL/6 mice were injected subcutaneously with B16F10 melanoma cells(0.5×10⁶) and tumors were allowed to form. Mice were next injected 3×per week with 10 ug per mouse of cPG peptide. Serum was collaged after14 days. Serum was diluted in assay buffer and the relative levels ofcytokines were examined by ELISA. FIG. 26A is a bar graph wherein databars represent relative levels of TGF-β as determined by ELISA fromcontrol (DMSO) treated mouse and two different cPG peptide treated mice.FIG. 26B is a bar graph wherein data bars represent relative levels ofIL-4 as determined by ELISA from control (DMSO) treated mouse and twodifferent cPG peptide treated mice. FIG. 26C is a bar graph wherein databars represent relative levels of IL-1β as determined by ELISA fromcontrol (DMSO) treated mouse and two different cPG peptide treated mice.Thus, these results demonstrate that injection of mice with a peptidecontaining the HU177 epitope itself can induce the expression of apotent immune suppressive molecule TGF-β.

Example 21: Mab HU177 Enhances the Anti-Tumor Activity of the ImmuneCheckpoint Inhibitor Anti-PDL-1 Antibody

Mice (C57BL/6) were injected with 3.5×10⁵ B16F10 melanoma cells. Micewere allowed establish pre-existing tumors for 5 days prior totreatment. Mice were treated (100 ug/mouse) with either anti-PD-L1antibody alone, Anti-HU177 antibody alone or a combination of bothantibodies 3 times a week for 15 days. FIG. 27A is a bar graph whereindata represents mean tumor volume at day 7+SE from 7 mice per condition.FIG. 27B is a bar graph wherein data represents mean tumor volume at day15+SE from 7 mice per condition. Thus, these results demonstrate thatmurine Mab HU177 directed to the HU177 cryptic collagen epitope canenhance the anti-tumor activity of the immune checkpoint inhibitoranti-PD-L1.

Example 22: Synthetic Collagen Peptides Containing the Amino AcidSequence RGD Support T Cell Adhesion

In order to demonstrate that T-cells can directly interact and bind tothe cryptic collagen epitopes recognized by Mab XL313 (i.e., P-2 andP-4), synthetic peptides containing amino acid sequences of collagentype-1 (P1 through P-5) along with a mutated control collagen peptide(P-C) were immobilized on non-tissue culture wells (250 ng/well). HumanT-cells (Jurkat) were added and allowed to attach to the collagenpeptides. The results are presented in FIG. 28. Data bars indicate meancell adhesion±standard deviation from triplicate wells. P-1(CKGDRGDAGPC; SEQ ID NO: 19); P-2 (CQGPRGDKGEC; SEQ ID NO: 6); P-3(CAGSRGDGGPC; SEQ ID NO: 7); P-4 (CQGIRGDKGEC; SEQ ID NO: 20); P-5(CRGPRGDQGPC; SEQ ID NO: 9); P-C(CQGPSGAPGEC; SEQ ID NO: 10).

Example 23: Monoclonal Antibody (Mab XL313) Inhibits InteractionsBetween Human T-Cells and the Cryptic Collagen Epitope Peptide P-2(CQGPRGDKGEC: SEQ ID NO: 6)

In order to demonstrate that Mab XL313 can inhibit human T-cellinteractions with the cryptic collagen epitope peptide P-2 (CQGPRGDKGEC;SEQ ID NO: 6), the P-2 peptide was immobilized (100 μg/well) onnon-tissue culture wells and human T-cells (Jurkat) were added in thepresence (50 μg/ml) or absence of Mab XL313 or control, antibodiesnormal mouse Ig (Ab Cont) and anti-integrin receptor (αvβ3) and allowedto attach to the collagen peptides. The results are presented in FIG.29. Data bars indicate mean cell adhesion±standard deviation fromtriplicate wells.

Example 24: The Integrin Receptor αvβ3 Dose Dependently Binds theCryptic Collagen Epitope Peptide P-2

In order to demonstrate that the integrin receptor αvβ3 can bind to thecryptic collagen epitope peptide P-2, which is recognized by Mab XL313,solid phase ELISA was carried out. The cryptic collagen peptide P-2 wasimmobilized (100 μg/ml) on microtiter wells and increasingconcentrations of recombinant integrin αvβ3 was added and integrinbinding was quantified. The results are presented in FIG. 30. Data barsindicate mean integrin binding±standard deviation from triplicate wells.

Example 25: Mab XL313 Inhibits Recombinant Integrin Receptor αvβ3Binding to the Cryptic Collagen Epitope Peptide P-2

In order to demonstrate that Mab XL313 specifically inhibits integrinreceptor αvβ3 binding to the cryptic collagen epitope peptide P-2, solidphase ELISA was carried out. The cryptic collagen peptide P-2 wasimmobilized (100 μg/ml) on microtiter wells and recombinant integrinαvβ3 was added and it was allowed to bind in the presence (50 μg/ml) orabsence of Mab X1313 or control antibody (Mab XL166 directed to othercollagen RGD peptides). The results are presented in FIG. 31. Data barsindicate mean integrin binding±standard deviation from triplicate wells.

Example 26: Reducing Expression of β3 Integrin Leads to ReducedExpression of PD-L1 in Human T-Cells

In order to confirm the functional role of integrin αvβ3 in regulatingthe levels of the immune suppressive molecule PD-L1, β3 integrinexpression was knocked down by shRNA in human T-cells. Whole celllysates from control transfected (Cont) and β3 integrin shRNAtransfected T-cells (β3 KD) were analyzed for expression of β3 integrin(β3 Int), PD-L1 and beta actin (β-Actin). Western blot analysisindicates clear reduction in the levels of PD-L1 in β3 integrin knockdown cells as compared to control. The results are presented in FIG. 32.

Example 27: Reducing Expression of β Integrin Prevents Stimulation ofPD-L1 by the XL313 Cryptic Collagen Peptide P2 in Human T-Cells

In order to confirm the functional role of integrin αvβ3 receptor inregulating the levels of the immune suppressive molecule PD-L1 by theXL313 cryptic collagen peptide P2, the expression of PD-L1 was examinedin β3 integrin knock down and control transfected T-cells. Whole celllysates from control transfected (Cont T-cells) and β3 integrin shRNAtransfected T-cells (β3/KD) that were stimulated with control peptide(PC) or the XL313 cryptic collagen peptide (P2) were analyzed by westernblot for expression of PD-L1. While P2 peptide enhanced expression ofPD-L1 in control transfected T-cells, P2 showed little ability toenhance the expression of PD-L1 in β3 integrin knock down cells (β3 KD).The results are presented in FIG. 33.

Example 28: Anti-XL313 Antibody Reverses the Inhibitory Effects of theCryptic Collagen Peptide P2 on T-Cell Migration

Membranes of transwell migration chambers were coated with denaturedcollagen type-IV to mimic the altered basement membrane in tumorvessels. The results are presented in FIG. 34. Left). T-cell (Jurkat)migration in the presence or absence of the cryptic collagen peptide P2(1.0 ug/ml) and/or Mab XL313 and non-specific control antibody (50μg/ml). Right). T-cell (Jurkat) migration in the presence or absence ofRaw macrophage concentrated conditioned medium (CM) and/or Mab XL313 andnon-specific control antibody (50 μg/ml). Data bars represent mean cellmigration ±SE from triplicate wells.

Example 29: The Cryptic Collagen Peptide P2 Induces Expression of theImmunosuppressive Molecule LAG3 in Human T-Cells

In order to confirm the functional ability of the XL313 cryptic collagenpeptide P2 to regulate expression of the immune suppressive checkpointmolecule LAG3, αvβ3 expressing human T-cell lines Jurkat and CCL120.1were stimulated with the cryptic collagen peptide P2 or control collagenpeptides P1 or PC. Whole cell lysates from were analyzed for expressionof PD-L1 and beta actin (β-Actin). Western blot analysis indicates clearincrease in the levels of PD-L1 in P2 stimulated T-cells as compared tocontrols. The results are presented in FIG. 35.

Example 30: Anti-XL313 Mab Reduces the Levels of the Immune SuppressiveMolecule LAG-3 in Melanoma Tumors In Vivo

C57BL/6 mice were injected with B16F10 melanoma cells (0.5×10⁶). Micewere treated (i.p) with anti-XL313 or control antibody (50 μg 3×/wks.).B16F10 tumors (N=3 from each group) were stained for expression of LAG-3(green). The results are presented in FIG. 36. Left). Examples of LAG-3expression from each condition. Photos were taken at 200× and 400×magnification. Right). Quantification (Image J) of mean±S.E relativeLAG-3 expression from 5 100× fields from each of 3 different tumors fromeach condition.

Example 31: Anti-XL313 Mab Enhances the Levels of CD8+ T-Cells inMelanoma Tumors in Vivo

C57BL/6 mice were injected with B16F10 melanoma cells (0.5×10⁶). Micewere treated (i.p) with anti-XL313 or control antibody (50 μg 3×/wks.).Single cell suspensions of whole tumors from each condition wereprepared and the levels of CD8+ T-cells from each tumor (N=4 per group)were quantified by flow cytometry. Data bars represent the mean level oftumor CD8+ T-cells ±SE from single cell suspensions from 4 tumors percondition. The results are presented in FIG. 37.

Example 32: Anti-HU177 Mab Reduces the Levels of Immune SuppressiveCD4+/CTLA-4 Positive T-Reg Cells in Melanoma Tumors In Vivo

C57BL/6 mice were injected with B16F10 melanoma cells (0.5×10⁶). Micewere untreated or treated (i.p) with anti-HU177 or control antibody (50μg 3×/wks.). Single cell suspensions of whole tumor from each conditionwere prepared and the levels of CD4+/CTLA-4 positive T-Reg cells fromeach tumor (N=4 per group) were quantified by flow cytometry. Theresults are presented in FIG. 38. Data bars represent the mean level ofT-Reg-cells ±SE from single cell suspensions from 4 tumors percondition.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

We claim:
 1. A method of treating cancer in a subject comprising,identifying a subject that has been diagnosed with cancer; administeringan immune checkpoint inhibitor; and administering an antagonist ofcollagen or a fragment thereof, thereby treating cancer in said subject.2. The method of claim 1, wherein the immune checkpoint inhibitorcomprises an inhibitor of CTLA-4, PD-1, PDL-1, Lag3, LAIR1, or LAIR2 3.The method of claim 1, wherein said inhibitor of PDL-1 comprises a PDL-1antibody.
 4. The method of claim 1, wherein said antagonist of collagenor a fragment thereof enhances anti-tumor activity of the immunecheckpoint inhibitor.
 5. The method of claim 1, further comprisinginhibiting an inflammatory condition, wherein said inflammatorycondition comprises dermatitis, pneumonitis, or colitis.
 6. The methodof claim 1, wherein the collagen comprises collagen type-I, collagentype II, collagen type III, or collagen type-IV.
 7. The method of claim6, wherein the antagonist of collagen type-IV or a fragment thereofcomprises an antagonist of the XL313 cryptic collagen epitope or anantagonist of the HU177 cryptic collagen epitope.
 8. The method of claim7, wherein the antagonist of the XL313 cryptic collagen epitopecomprises an antibody that binds a cryptic RGDKGE (SEQ ID NO:1)-containing collagen epitope or wherein the antagonist of the HU177cryptic collagen epitope comprises an antibody that binds a crypticCPGFPGFC (SEQ ID NO: 16)-containing collagen epitope.
 9. The method ofclaim 8, wherein the antibody comprises a monoclonal antibody.
 10. Themethod of claim 9, wherein said monoclonal antibody comprises an XL313monoclonal antibody or an HU177 monoclonal antibody.
 11. The method ofclaim 1, wherein the subject is a human.
 12. The method of claim 1,further comprising administering a chemotherapy agent.
 13. The method ofclaim 1, wherein the cancer is selected from the group comprising ofmelanoma, central nervous system (CNS) cancer, CNS germ cell tumor, lungcancer, leukemia, multiple myeloma, renal cancer, malignant glioma,medulloblatoma, breast cancer, ovarian cancer, prostate cancer, bladdercancer, fibrosarcoma, pancreatic cancer, gastric cancer, head and neckcancer, colorectal cancer. For example, a cancer cell is derived from asolid cancer or hematological cancer. The hematological cancer is, e.g.,a leukemia or a lymphoma. A leukemia is acute lymphoblastic leukemia(ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia(CML), or acute monocytic leukemia (AMoL). A lymphoma is follicularlymphoma, Hodgkin's lymphoma (e.g., Nodular sclerosing subtype,mixed-cellularity subtype, lymphocyte-rich subtype, or lymphocytedepleted subtype), or Non-Hodgkin's lymphoma. Exemplary solid cancersinclude but are not limited to melanoma (e.g., unresectable, metastaticmelanoma), renal cancer (e.g., renal cell carcinoma), prostate cancer(e.g., metastatic castration resistant prostate cancer), ovarian cancer(e.g., epithelial ovarian cancer, such as metastatic epithelial ovariancancer), breast cancer (e.g., triple negative breast cancer), and lungcancer (e.g., non-small cell lung cancer).
 14. The method of claim 1,wherein said immune checkpoint inhibitor is administered at a dose of0.01-10 mg/kg bodyweight, and wherein said antagonist of collagen isadministered at a dose of 0.01-25 mg/kg bodyweight.
 15. The method ofclaim 1, wherein said composition is administered once per hour, orwherein said composition is administered once every two weeks for 4 to 6weeks.
 16. A method of treating a disease characterized by abnormalimmune suppression in a subject comprising, identifying a subject thathas been diagnosed with a disease characterized by abnormal immunesuppression; administering an immune checkpoint inhibitor; andadministering an antagonist of an integrin, thereby treating in saidsubject.
 17. The method of claim 16, wherein the immune checkpointinhibitor comprises an inhibitor of CTLA-4, PD-1, PDL-1, Lag3, LAIR1, orLAIR2.
 18. The method of claim 16, wherein said immune checkpointinhibitor comprises a CTLA-4 antibody, a PD-1 antibody, a PDL-1antibody, a Lag3 antibody, a LAIR1 antibody or a LAIR2 antibody.
 19. Themethod of claim 18, wherein the integrin comprises integrin αvβ3. 20.The method of claim 19, wherein said antagonist of integrin αvβ3comprises an antibody targeting αvβ3 binding RGDKGE (SEQ ID NO:1)-containing collagen epitope.
 21. The method of claim 18, wherein theintegrin comprises integrin α10β1.
 22. The method of claim 21, whereinsaid antagonist of integrin α10β1 comprises an antibody targeting α10β1binding CPGFPGFC (SEQ ID NO: 16)-containing collagen epitope.
 23. Themethod of claim 16, wherein the subject is a human.
 24. The method ofclaim 16, further comprising administering a chemotherapy agent.
 25. Themethod of claim 16, wherein the disease characterized by abnormal immunesuppression comprises Type I diabetes, lupus, psoriasis, scleroderma,hemolytic anemia, vasculitis, Graves' disease, rheumatoid arthritis,multiple sclerosis, Hashimoto's thyroiditis, Myasthenia gravis, andvasculitis.
 26. The method of claim 16, wherein said immune checkpointinhibitor is administered at a dose of 0.01-10 mg/kg bodyweight, andwherein said antagonist of an integrin is administered at a dose of0.01-25 mg/kg bodyweight.
 27. The method of claim 16, wherein saidcomposition is administered once per hour, or wherein said compositionis administered once every two weeks for 4 to 6 weeks.
 28. A method oftreating a disease characterized by an overactive immune response in asubject comprising, identifying a subject that has been diagnosed withan overactive immune response; and administering a peptide comprisingcollagen or a fragment thereof, thereby treating an overactive immuneresponse in said subject.
 29. The method of claim 28, wherein saidoveractive immune response comprises an autoimmune disease.
 30. Themethod of claim 28, wherein the collagen comprises collagen type-I,collagen type II, collagen type III, or collagen type-IV.
 31. The methodof claim 28, wherein the peptide comprises RGDKGE (SEQ ID NO: 1) orCPGFPGFC (SEQ ID NO: 16).
 32. The method of claim 28, wherein thesubject is a human.
 33. The method of claim 28, wherein said autoimmunedisease comprises Graves' disease, Hashimoto's thyroiditis, Systemiclups erythematosus (lupus), Type 1 diabetes, multiple sclerosis orrheumatoid arthritis.
 34. A method for healing a wound in a subjectcomprising, identifying a subject with a wound; and administering apeptide comprising collagen or a fragment thereof to said wound, therebyhealing said wound in said subject.
 35. The method of claim 34, whereinthe collagen comprises collagen type-I, collagen type II, collagen typeIII, or collagen type-IV.
 36. The method of claim 34, wherein thepeptide comprises RGDKGE (SEQ ID NO: 1) or CPGFPGFC (SEQ ID NO: 16). 37.The method of claim 34, wherein the subject is a human.